ACID Phosphatase1

ACID PHOSPHATASE' Oscar Bodansky Sloan-Kettering Institute for Cancer Research. New York. N e w York

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Introduction .. 2.1.

Activity . . . . . . . . . . . . . . . . . .

Introduction .

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The Bodansky Method .......................................... The p-Nitrophenyl Phosphate Method . . . . . . . . . . . . . . Method of Huggins and Talalay .................................. 8-Naphthyl Phosphate Method . . . . . . . . . . ....... a-Naphthyl Phosphate Method ................................... Comparison of Acid Phosphatase Activities Determined by Different Methods ....................................................... 2.9. Current Methods for Determination of Serum Acid Phosphatase Activity ......................................................... 2.10. Determination of Acid Phosphatase Activity in Blood Cells and in Tissues ........................................................ Acid Phosphatases from Different Tissues: Purification, Isoeneymes, and Propertpies........................................................... 3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Human Prostatic Acid Phosphatase ............................... 3.3. Human Erythrocytic Acid Phosphatase ............................ 3.4. Human Leukocytic Acid Phosphataae ............................. 3.5. Liver Acid Phosphatase ......................................... 3.6. Spleen Acid Phosphatase ........................................ 3.7. Human Placental Acid Phosphatase . . . . . . . . . . . . . Intracellular Distribution of Acid Phosphatase ........................... 4.1. Introduction ...................... .......................... 4.2. Intracellular Distribution of Acid Phosphatase in Liver .............. 4.3. Intracellular Distribution of Acid Phosphatase in Other Tissues . . . . . . 4.4. Digestive Function of Lysosomes ................................. Polymorphism of Acid Phosphatase in Human Erythrocytes . . . . . . . . . . . . . . . 5.1. Introduction ....................... ......................... 5.2. Electrophoresis ................................................. 5.3. Genetics ....................................................... 5.4. Quantitative Distribution . . . . . . . . . . . . . . .................... 5.5. Biochemical Characteristics of Phenotypes ......................... 5.6. Polymorphism in Other Tissues ................................... Alterations of Serum Acid Phosphatase Activity in Disease . . . . . . . . . . . . . . . . 6.1. Introduction ................................................... 6.2. Normal Values for Serum Acid Phosphatase Activity . . . . . . . . . . . . . . . . 2.3. 2.4. 2.5. 2.6. 2.7. 2.8.

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'This work was supported in part by Grant CA-08748 from the National Cancer Institute. National Institutes of Health 43

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Serum Acid Phosphatase in Carcinoma of the Prostate.. ............ Specificity of Serum Acid Phosphatase Determination for Carcinoma of the Prostate.. ............................................... 6.5. Facton Involved in Elevation of Serum Acid Phosphatase in Carcinoma of the Prostate.. ............................................... 6.6. Acid Phosphatase Activity in Nonprostatic Disease. . . . . . . . . . . . . . . . . 6.7. Serum and Plasma Acid Phosphatase Activity in Hematologic and Hematopoietic Disease. ......................................... 6.8. Acid Phosphatase Activity in Gaucher’s Disease. . . . . . . . . . . . . . . . . . . . 6.9. Leukocytic Acid Phosphatase Activity in Hematologic and Hematopoietic Disease.. ............................................... 6.10. Serum Acid Phosphatme in Thromboembolism. .................... 6.11. Serum Acid Phosphatase in Diseases of Childhood.. ................ 7. Lysosomal Disease and Acid Phosphatase Activity.. ...................... 7.1. Introduction ................................................... 7.2. Lysosomes and Cancer. ~........................................ 7.3. Deficiency of Lysosomal Acid Phosphatase.. ....................... 7.4. Multiple Lysosomsl Enzyme Deficiency. .......................... 7.5. Hemorrhagic Enteropathy and Lysosomal Enzymes. ................ References............................................................... 6.3. 6.4.

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Introduction

The existence of the enzyme acid phosphatase. was first revealed in 1925 when Demuth (D11) observed that human urine was capable of hydrolyzing hexose diphosphate with optimal activity occurring a t a pH of approximately 5.0. In 1935 and 1936, Kutscher and his associates (K12, K13) found that this enzyme was present to some extent in the human testis, epididymis, seminal vesicle, spermatic cord and, in a remarkably high concentration, in the prostate. Within the next few years, Gutman and his associates (G10, G11, G12, G13, G14) determined that this enzyme activity was present in serum and could be utilized as an indicator of the presence of carcinoma of the prostate. Since these early discoveries, this enzyme has assumed additional biological and medical significance. I n 1955, de Duve and his associates described the association of acid phosphatase with “a special class of cytoplasmic granule” in rat liver (A13, DlO), and this enzyme subsequently became the marker for a new intracellular component, the lysosome. Recently, lysosomal acid phosphatase deficiency has been described in man ( N l ) . As the study of acid phosphatase progressed, increasing indications arose that there might be differences among the acid phosphatases of different tissues (A4, D13, K3), and more recently, the activities of acid phosphatases in platelets ( Z l ) , in normal and abnormal leukocytes (B8, L7, L8, V l ) , and in Gaucher cells (L8) have been described to be indicators of corresponding pathological states. I n addition, it has been shown that the acid phosphatase within a given tissue may

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consist of more than one molecular form, or isoenzyme (L14, L15, S24, S31), and this finding has had genetic (H11) and pathophysiological implications. And even with regards to the earliest utilization of acid phosphatase, its determination in the serum as an indicator of the presence of carcinoma of the prostate, methodological advances and increasing clinical biochemical correlations have tended to define this role more precisely. In view of these various considerations, it seemed most appropriate to review the various aspects of this subject for readers of these Advances, particularly since no review has appeared since the inception of the series in 1958. 2.

Methods of Determination

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Acid Phosphatase Activity

2.1. INTRODUCTION

A considerable number of procedures have been utilized to assay the acid phosphatase activity of serum, blood cells, and tissues. These have involved different substrates or concentrations of substrates, different temperatures, buffers, or variations in other conditions. If the same acid phosphatase were being measured, then the results were naturally not comparable. But the possibility also exists that closely related but different acid phosphatases were present within the same tissue or in different tissues, and the rate of action of these acid phosphatases depended on the particular substrates, buffers that were employed, or other conditions of the reaction. It seems most appropriate then to preface our review and consideration of the literature by describing briefly the conditions characterizing the most frequently used procedures for the determination of acid phosphatase activity, particularly in the serum. Other methods, or modifications of those t o be presented here, will be described in later sections of this review. 2.2. THE GUTMANMETHOD(G10, G14)

This was the first method used in assaying serum acid phosphatase activity and was a modification of the King-Armstrong (K4) method for alkaline phosphatase. The buff er-substrate was 0.005 M disodium monophenyl phosphate in Sorensen's 0.1 M citrate buffer adjusted to p H 4.8. To 10 ml of this mixture, brought to 37"C, 0.5 ml of serum a t 37°C was added, yielding a pH of 4.9. The contents were stirred and allowed to incubate for 3 hours ; the liberated phenol was determined. The activity was defined in units, as the number of milligrams of phenol liberated in 1 hour a t 37°C by 100 ml of serum. For serums of high activity, shorter times of incubation or dilutions of serum were used. The normal range

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was stated as lying between 0 and 2.5 units, but this will be considered more fully later (Section 6.2). Shortly after publication, this procedure was utilized by several investigators with only minor changes, such as in the p H of the citrate buffer (H7, W l ) the use of a sodium acetate-acetic acid buffer at pH 5.0 (H17) or, subsequently, the determination of hydrolyzed phenol with aminoantipyrine (K4). The concentration of phenyl phosphate, before the addition of serum, was 0.005 M in all these instances. Since the innovation of Gutman and Gutman (G10, G14) consisted in adapting the use of the phenyl phosphate in the King-Armstrong method for alkaline phosphatase to the determination of acid phosphatase a t pH 5.0, the procedure will be referred to in this paper as the Gutman method. 2.3. THEBODANSKY METHOD(B18, 52)

This procedure was based on the use of P-glycerophosphate as substrate as in A. Bodansky's method for alkaline phosphatase (B17). In this method the mixture, to which the serum was added to start the reaction, had concentrations of 0.016 M sodium P-glycerophosphate and 0.021 M sodium diethylbarbiturate. The addition of 1.0 ml serum t o 10 ml of this mixture or, as in a later version, of 0.5 ml to 4.5 ml of the mixture led to concentrations in the final reaction mixture of 0.0144 M sodium P-glycerophosphate and 0.019 barbiturate buffer. In the first brief description of his procedure for acid phosphatase, A. Bodansky incorporated acetic acid in the P-glycerophosphate-diethylbarbiturate mixture so as to bring it to pH 5.0 before adding serum (1 volume to 9 volumes of mixture) to start the reaction. This proceeded for a period of 3 hours a t 37"C, when it was terminated by the addition of trichloroacetic acid. The acid phosphatase activity was expressed as the number of milligrams of inorganic phosphorus liberated as phosphate in 1 hour by 100 ml of serum under the conditions of this assay. The normal range of acid phosphatase values was defined as lying between 0.1 and 0.4 unit (52). In a later version (BlS), the diethylbarbiturate buffer was omitted; to 6.93 ml of unbuffered 0.016M sodium P-glycerophosphate, 0.27 ml of 0.50 N hydrochloric acid was added. The mixture was warmed to 37.5"C, and 0.8 ml of serum also warmed to 37.5"C was added. The mixture was allowed to incubate for 2 hours, when the reaction was stopped by the addition of trichloroacetic acid. The acid phosphatase activity was expressed in units as before. The average activity in 43 males was 0.19 unit with a standard deviation of 0.048 unit (€319). It is of interest in this connection that, in the course of studying a patient with prostatic carcinoma and extensive prostatic calcification, Barringer

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and Woodard (B5) determined the action of serum on P-glycerophosphate over a range of pH levels from 6.0 to 9.0 and found the ratio of activity a t pH 6.4 to that of 9.0 elevated in some cases of prostatic carcinoma. However, no definite method for serum acid phosphatase activity was proposed a t this time. Shinowara et al. (S18) made a slight modification in the Bodansky procedure. To 50 ml of stock substrate solution containing 0.0317M sodium P-glycerophosphate and 0.0412 M sodium diethylbarbiturate, 5 ml of 1.0 M acetic acid was added, and the solution made up to 100 ml. The pH was 5.0 or was adjusted to this pH. One volume of serum, undiluted or diluted to various strengths, was added to 10 volumes of the diluted substrate, and the reaction was allowed to proceed for 1 hour when it was stopped by the addition of trichloroacetic acid. The concentrations in the reaction mixture, which had a pH of 5.0, were 0.0144M sodium P-glycerophosphate, 0.0185 M diethylbarbiturate, and 0.045 M acetate. The units were expressed as in the Bodansky method, namely, milligrams of phosphorus liberated per 100 ml of serum in 1 hour, and the range was 0.0-1.1 units in 20 healthy subjects and in 140 control patients. 2.4. THEp-NITROPHENYL PHOSPHATE METHOD

The relative rates of hydrolysis of various phosphate esters, including p-nitrophenyl phosphate, a t alkaline pH levels ranging from 8.08 to 9.80, were studied by King and Delory in 1939 (K5a). In 1937 Ohmori (01) had investigated the hydrolysis of p-nitrophenyl phosphate a t p H levels ranging from 2.0 to 9.0 by various “phosphatase” preparations from pig kidney, dried yeast, guinea pig blood, and “takaphosphatase.” He noticed that several of these preparations showed optimal activities in the acid region, a t about pH 4.0-5.0. In 1947, Hudson et al. (H15) developed a method for acid phosphatase which, like the procedure of Bessey et al. for alkaline phosphatase (B16) , was based upon the use of p-nitrophenyl phosphate as substrate. The buffer substrate solution consisted of equal volumes of a 0.1M sodium acetate-acetic acid buffer, pH 5.4, and 0.001 M magnesium chloride and of a 0.4% solution of approximately 50% pure disodium p-nitrophenyl phosphate in 0.001 N HC1. To 1 ml of this solution, 0.1 ml of the serum sample was added. The final concentrations in this reaction mixture were 0.045M acetate buffer, pH 5.4; magnesium chloride. 0.00045M ; substrate, 0.004 M . The reaction was allowed to run for 30 minutes a t 38”C, and the reaction was stopped by the addition of sodium hydroxide. The liberated yellow p-nitrophenol was read at 400 nm and the amount was

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calculated from a suitable calibration curve. The units of acid phosphatase activity were defined as the number of millimoles which were liberated in 1 hour by 1 liter of serum. In 47 normal individuals, the mean value was 1.54 units with a standard deviation of 0.34 unit.

2.5. METHODOF HUGGINS AND TALALAY (H18) The principle of this method consists in the action of phosphatase, whether alkaline or acid, on phenolphthalein diphosphate to liberate phenolphthalein which, a t alkaline pH, is pink or red. The intensity of the color was measured immediately, and the amount of phenolphthalein was determined from a suitable calibration curve. Huggins and Talalay (H18) synthesized their substrate and obtained a preparation which they believed to be sufficiently pure for their purposes. The procedure for the determination of acid phosphatase activity was as follows. To 5 ml of a solution containing about 0.001M sodium phenolphthalein diphosphate, dissolved in 0.1M acetic acid-acetate buffer of pH 5.4 and warmed to 37"C,0.5 ml of serum or of another of acid phosphatase, also warmed to 37"C,was added. The contents were mixed, and the mixture was incubated for precisely 1 hour a t 37°C;4.5ml of an alkaline glycine buffer, pH 11.2,was then added to stop the reaction; the color was read immediately. The units were expressed as the number of milligrams of phenolphthalein liberated by 100 ml of serum under the stated conditions. The serum acid phosphatase levels for 41 normal males, aged 21 to 65, and 15 normal females, aged 21 to 50,gave an average value of 5.9 units with a range of 3 to 10 units. No difference between the sexes was observed. 2.6. p-NAPHTHYL PHOSPHATE METHOD In 1950 Seligman and his co-workers (S13)suggested the use of sodium P-naphthyl phosphate as a substrate for the determination of acid or alkaline phosphatase activity. For the former, 1 ml of 1 :20 diluted serum was added to 5 ml of 0.4 mM sodium p-naphthyl phosphate in 0.1M acetate buffer of pH 4.8,and the reaction was allowed to proceed for 2 hours a t 37.5"C. The addition of 4 drops of 1 M sodium carbonate solution served to retard the reaction as well as to raise the p H to the optimal level for coupling with 1 ml of a solution of tetrazotized orthodianisidine. After 3 minutes, the protein was precipitated with trichloroacetic acid, the dye extracted with ethyl acetate, and the color density determined in the region of 540 nm. The unit of phosphatase activity was defined as that amount of enzyme which liberates the color equivalent of 10 ml of p-naphthol per hour a t 37.5" in 1 hour. The serum acid phosphatase in a group of normal adults ranged from 0.7 to 1.6 units and averaged 1.0 unit per 100 ml of serum.

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2.7. a-NAPHTHYL PHOSPHATE METHOD Babson et al. ( B l , B2) introduced the use of this substrate with the suggestion that it was highly specific for the presence of prostatic acid phosphatase in the serum. The substrate-buffer mixture is a commercially designed tablet containing 0.67 mg of sodium a-naphthyl acid phosphate in a mixture of citrates designed to yield a pH of 5.2 in the reaction mixture. To this tablet, dissolved in 0.5 ml of water and warmed to 37"C, 0.2 ml of serum was added and the reaction was allowed to incubate a t 37°C for precisely 30 minutes. The mixture was cooled a t 15-20°C, and a tablet containing 0.4 mg of tetrazotized orthodianisidine in a stabilized form was added and crushed with a glass rod. The solution was diluted to 5.0, and the optical density of the resulting colored solution a t 530 nm was read exactly 3 minutes after the addition of the color developer. Suitable controls are employed. The unit of acid phosphatase was defined as the amount of enzyme that will liberate 1 mg of a-naphthol per hour. The activities of serum acid phosphatase in 56 apparently normal, healthy young men ranged from 0.9 to 5.5 units per 100 ml of serum and yielded a mean value of 2.0 f 0.7 units. The activities in 33 apparently normal women ranged from 0.5 to 2.6 units and gave a mean value of 1.5 Ifr 0.5 units. Babson et al. (B2) believed that this method was highly specific for the prostatic component of serum acid phosphatase. They determined the activities of mixtures of heated serum with prostatic acid phosphatase or erythrocytic acid phosphatase on the series of substrates used in various methods. The ratio of activities was designated as the relative specificity for prostatic acid phosphatase and had the following values: phenyl phosphate, 2.3 ; phenolphthalein phosphate, 0.9; p-nitrophenyl phosphate, 1.2; /3-naphthyl phosphate, 1.9; P-glycerophosphate, 48; a-naphthyl phosphate, 98. Thus, it would appear that the a-naphthyl phosphate is the most specific procedure for the determination of the presence of prostatic acid phosphatase. However, it should be noted that the assumption underlying the work of Babson e t al. (B2) is that there are only two types of acid phosphatase in the serum, and it is quite possible that there are several others. I n a study of 120 patients without cancer and 87 with prostatic cancer, Seal e t al. (512) found that the a-naphthyl phosphate substrate method was as sensitive or more sensitive than the tartrate-inhibitable phenyl phosphate substrate method.

2.8. COMPARISON OF ACIDPHOSPHATASE ACTIVITIES DETERMINED BY DIFFERENT METHODS

It is apparent from the preceding discussion that the rate of action of the acid phosphatase present in normal serum varies with the par-

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ticular organic phosphate compound used as substrate and its concentration in the reaction mixture. Since the “acid phosphatase” in the serum is undoubtedly a mixture of the enzyme from various tissues, it would be irrelevant to carry out any precise kinetic studies at different concentrations of each substrate and thus determine Michaelis constants. The normal average values for the activities that have been noted above by the various methods may all be converted into micromoles of substrate hydrolyzed in 1 hour a t 37-38°C by 100 ml of serum to yield the following comparison: P-glycerophosphate, 6.1 ; phenyl phosphate, 15; phenolphthalein diphosphate, 18; p-nitrophenyl phosphate, 145 ; P-naphthyl phosphate, 69; a-naphthyl phosphate, 14. As will be illustrated subsequently, the relative rates of action on the different substrates may differ even more widely in patients with elevated serum acid phosphatase activities arising by the admixture of acid phosphatases from different tissues. As the preceding considerations illustrate and as was noted a t the beginning of this section (2.1), comparison of acid phosphatase activities obtained in different studies must take into account the method employed. Some workers have attempted to do this by using the terms “P-glycerophosphatase,” “phenylphosphatase,” etc. to designate the substrate employed (B6, T6). However, such usage may imply that different “acid phosphatases” are responsible for these actions, and we shall therefore attempt to avoid this usage in the present review. 2.9. CURRENT METHODS FOR DETERMINATION OF SERUM ACIDPHOSPHATASE ACTIVITY Most of the methods that are currently being employed in clinical laboratories or in investigations are either those that have just been described or are slight modifications thereof. For example, Linhardt and Walter (L9) have chosen for inclusion in Bergmeyer’s “Methods of Enzymatic Analysis” (L9), the procedures of Huggins and Talalay (H18) utilizing phenolphthalein diphosphate as substrate and of Hudson et al. (H15) with p-nitrophenyl phosphate as substrate. The writer instituted the method of A. Bodansky (B18, 52) in the Department of Biochemistry, Memorial Hospital, New York in 1948, and this method is still being employed there. Levinson’s and MacFate’s text “Clinical Laboratory Diagnosis” (L6) has selected the method described by A. Bodansky (B18, 52) as modified by Shinowara et a2. (518). “Bray’s Clinical Laboratory Method” (B7) has chosen the procedure of Hudson et al. (H15) except that citrate-citric acid buffer, pH 5.0, is substituted for the acetate-acetic acid buffer, pH 5.0. Automated methods based on

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the manual methods that have been described above are also coming into use (G9, K9). The question may arise as to which is the preferred method. I n the author’s experience, and this will be documented more completely later, the use of the substrate, sodium P-glycerophosphate, as in the Bodansky procedure (B18, 521, is more specific for elevations of serum acid phosphatase activity due to prostatic carcinoma. However. the use of other substrates, such as sodium phenyl phosphate in the Gutman method (G10, G14), may elicit alterations of activity in the serum that reflect diseases in other tissues. 2.10. DETERMINATION OF ACIDPHOSPHATASE ACTIVITYIN BLOOD CELLSAND I N TISSUES

As we have seen, practically all the methods on the determination of acid phosphatase activity in serum are calculated upon the amount of reaction product, such as inorganic phosphate, phenolphthalein, or p-nitrophenol, that would be produced under the conditions stated for the method by 100 ml of serum or, as in the method of Hudson e t al. (H15), by 1 liter of serum. In the case of the acid phosphatase activity of tissues, some other basis for calculation is used, although the method may be the same as that used for serum. A few examples will be cited here in illustration. I n their study of the properties of acid phosphatases of erythrocytes and of the human prostate gland, Abul-Fad1 and King ( A 4 ) employed a substrate-buffer mixture consisting of equal volumes of 0.02 .M disodium phenyl phosphate and of acetate buffer (concentration not stated). The volume of hemolysate or of prostatic gland extract that was added to this mixture was not stated, and the reaction was allowed to proceed for exactly 30 minutes at 37°C. The activities were expressed as milligrams of phenol liberated per milliliter of red cells per hour or as milligrams of phosphate liberated in 30 minutes per 100 ml of enzyme solution. The red cell preparation was presumably a 1: 10 hemolysate, but the precise dilution of the prostatic preparation was not given. Woodard (W6, W8) employed the method of A. Bodansky (B18, 52) in determining the acid phosphatase activity of various human tissues. She calculated her activities as the number of milligrams of phosphorus that would be liberated per hour by 1 g of tissue under the defined conditions of the assay. In assaying the distribution of acid phosphatase in the rat ventral prostate, Bertini and Brandes (B15) employed a total reaction volume of 0.40 ml containing 0.28 M sodium glycerophosphate (it was not stated whether this was or p ) in 0.05 M acetate buffer. Results were expressed as micro(Y

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grams of liberated phosphorus after 10 minutes' incubation a t 38"C, and calculated per gram of wet tissues. I n studying the intracellular distribution of acid phosphatase in rat liver, de Duve and his associates (A12, A13, D9, D10, G2) measured the amount of inorganic phosphate liberated a t 37°C in the presence of 0.05 M P-glycerophosphate and 0.05 M acetate, adjusted to p H 5.0, and expressed the activities as micrograms of P liberated in 10 minutes a t 37°C per gram of liver. I n studies of the acid phosphatase activity of leukocytes in normal persons and in patients with leukemia or other blood dyscrasias, the activities were expressed as milligfams of phosphorus liberated in 1 hour by 1Olo cells from a reaction mixture at pH 5.0 containing a final concentration of 0.02M sodium P-glycerophosphate as substrate (B8, B9, V l ) . 3.

Acid Phosphatases from Different Tissues: Purification, Isoenzymes, and Properties

3.1. INTRODUCTION Study of the distribution of acid phosphatase in different tissues is burdened by indications that there are several acid phosphatases. Even the older literature indicated the nonidentity of acid phosphatases of different origin. I n 1934, Davies (D4) showed that the acid phosphatase in the red cell hydrolyzed a-glycerophosphate more readily than P-glycerophosphate, whereas the reverse was true for the acid phosphatase from spleen. Kutscher and Wolbergs (K12) found that prostatic acid phosphatase was inactivated irreversibly by various narcotics, including alcohols. A more systematic study of the acid phosphatases of erythrocytes and of human prostate was undertaken in 1949 by Abul-Fad1 and King (A4). The preparations were crude, the prostatic phosphatase being obtained by grinding human prostate with a 5-fold volume of 0.9% NaCl. The erythrocytic phosphatase consisted of centrifuged red cells, separated from white cells, washed twice with 0.9% NaCl and hemolyzed in 9 volumes of water. The buffer-substrate mixture consisted of equal volumes of acetate buffer (concentration not stated) and 0.02 M disodium phenyl phosphate. The erythrocytic acid phosphatase from man and several other species showed two pH optima, one a t a range of pH 4.3-4.8 and the second a t pH 5.S5.7. A concentration of 0.01 M Mg2+inhibited these activities to the extent of about 30-50.7'0 a t the lower p H levels and somewhat less so in the region of the higher pH optimum. Human prostatic acid phosphatase showed one clear pH optimum, a t about 5.0-5.2, and the inhibition by 0.01 M Mg2+was about 30% in this region.

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The relative rates of hydrolysis of several substrates were determined a t 37°C and pH 5.0 and expressed as milligrams of phosphorus liberated in 30 minutes per 100 ml of a diluted enzyme preparation. For 0.02M P-glycerophosphate in the absence of any added Mg2+these rates were 0.2 for erythrocytic phosphatase and 29 for prostatic phosphatase. The corresponding rates were 11 and 28 with 0.02M a-glycerophosphate as substrate, and 55 and 53 with O . 0 5 M phenyl phosphate as substrate. The presence of Mg2+ activated the rates of hydrolysis to only a small degree. Thus, it may be seen that the use of P-glycerophosphate as substrate distinguished sharply between the erythrocytic and prostatic phosphatases. Abul-Fad1 and King (All A2, A3, A4) also investigated the effect of various ions and organic compounds on the acid phosphatase activity of these two tissues. Without describing the results in detail, some of the outstanding effects may be noted. A concentration of 0.5 X 10-3M Cu2+ inhibited erythrocytic phosphatase to the extent of 8%96%, but prostatic phosphatase only to the extent of 1&18%. Similarly, 0.5% formaldehyde inhibited completely the erythrocytic phosphatase, but had no effect on prostatic phosphatase. The reverse patterns were shown by 0.5 X M FeS+(ferric) ion, which inhibited erythrocytic phosphatase slightly, about 5-976, and inhibited the prostatic enzyme to the extent of 80%. Fluoride in 0.01 M concentration also had comparatively little effect (8% inhibition) on erythrocytic phosphatase but exerted a marked inhibition, 96%, on prostatic phosphate. Of various organic radicals tested, only L-( )-tartrate (0.01 M ) had a marked differential effect, with 94% inhibition of the prostatic phosphatase and none of the erythrocytic phosphatase. These results were among the first to indicate the diverse nature of acid phosphatases from different sources and were the forerunner of other studies designed to differentiate among the acid phosphatases from different tissues as well as procedures aiming to determine the tissue source of elevations of this enzyme in the serum. An approximate idea of the distribution of acid phosphatase activity in human tissues, regardless of the nature of the acid phosphatase, may be obtained from the studies of Reis (R2) on 5'-nucleotidase and other phosphomonoesterases. He prepared aqeuous homogenates of postmortem tissue in the proportion of 20 parts of water to one of tissue, allowed these to autolyze for 2 days a t room temperature, centrifuged the material, and employed the supernatant fluid. The assay mixture consisted of 0.4 ml of a suitable buffer, 0.1 ml of 0.005M phenyl phosphate as substrate, and 0.1 ml of tissue extract. The enzyme activity was expressed as micrograms of phosphorus hydrolyzed per hour per milligram of wet

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weight tissue. At pH 5.5, the following activities were obtained: thyroid, 0.22; testicle, 0.30; media of aorta wall, 0.9; brain cortex, 0.5; optic nerve, 0.3; pituitary posterior lobe, 0.6; pituitary anterior lobe, 0.7; liver, 0.3; lung, 0.4; kidney medulla, 0.9; kidney cortex, 1.9; ossifying cartilage, 0.2; duodenal mucosa, 0.1 ; jejeunal mucosa, 0.4; prostate, 1030. The slight but definite elevations of serum acid phosphatase activity in conditions such as thrombocytopenia (02, Z l ) , Gaucher’s disease (T6, T8), or various myeloproliferative diseases (B6) indicate the possibility that platelets, the marrow, and the reticuloendothelial system may also be sources of acid phosphatase. These aspects will be discussed more fully later in the review. 3.2. HUMAN PROSTATIC ACIDPHOSPHATASE 3.2.1. Introduction The purification of acid phosphatase from the human prostate was undertaken, and high degrees of purity were obtained, before any solid information was available concerning the intracellular distribution of this enzyme or its existence in multiple molecular forms or isoenzymes. Accordingly, in this review several methods of purification will be described first, and the other aspects will then be considered. 3.2.2. Purification of Human Prostatic Acid Phosphatase A few of the outstanding contributions in this area will be briefly described. London and Hudson (L10) began their purification by slicing frozen human prostates into slices 0.5-1 mm in thickness, and adding to these slices 3 g of 0.2 M acetate buffer, pH 5.0, for each gram of tissue. The mixture was allowed to extract for 48-72 hours in the refrigerator with occasional shaking and then strained through cheesecloth. The tissue residue was extracted twice more in the same manner; the three extractions yielded about 80% of the activity originally present in the prostatic tissue. The combined extracts, which represented a 22-fold purification from the prostate, were dialyzed for 24 hours against distilled water a t room temperature. The material inside the cellophane bag separated into a precipitate, which accounted for 60-70% of the total protein, and a clear supernatant containing over 85% of the activity and representing a 41-fold purification. This supernatant was then mixed with calcium phosphate gel a t pH 7.5 and filtered. The filter cake was eluted with 0.02 M sodium citrate at pH 7.0, and the cake was washed with distilled water. The combined eluate, which showed an 81-fold purification, was concentrated by lyophilization. The enzyme solution was

ACID PHOSPHATASE

55

adjusted to pH 5.8 with 0.2 M acetic acid cooled to 0°C and fractionated rapidly with acetone a t 0”. The sediments from 36% acetone were discarded, and the sediments from 44% acetone were redissolved in half the starting volumes of distilled water. Treatment of this enzyme solution with ammonium sulfate between 60% and 68% saturation for 24 hours in the refrigerator resulted in a precipitate that represented 27% and a 296-fold purification of the enzyme activity in the original prostatic tissue. This material was dissolved in acetate buffer, diluted to 0.05% protein, placed in a gas washing bottle and caused to foam by passing in COz. The foam which contained almost all the protein was led off through the side arm. The remaining liquid or “frothate” was lyophilized and dialyzed against acetate buffers. Further concentration could be achieved by blowing hot air over the solution. The concentrated “frothate” amounted to 21% yield of the acid phosphatase originally present in the prostatic tissue and represented a 4900-fold purification. However, this preparation was unstable and even when kept in the refrigerator lost about 50% of its activity each month. I n 1958 Boman (B24) described a method of purification in two steps. The first one consisted of an extraction and a dialysis, and the second was a chromatographic fractionation. The starting material was human prostatic tissue and was stored at -15°C. The frozen prostatic tissue was cut into thin slices, weighed, and extracted with 5-fold its volume of 0.01% solution of Tween in cold distilled water a t 4°C. After 2 4 4 8 hours, the pink opalescent extract was filtered through glass wool and dialyzed against distilled water for about 3 days. The precipitate formed during the dialysis was removed through centrifugation and discarded. The brownish pink supernatant was freeze dried. Of this material (fraction 1) , 600 ml was dissolved in about 3 ml of McIlvaine’s citrate phosphate buffer of pH 5.50 and was dialyzed against this buffer for 6-12 hours. This solution was then applied to a column (72 X 3.3 cm) of Dowex 50 X-2 which had been equilibrated with the citrate phosphate buffer (B23). Elution was carried out successively with a citrate phosphate buffer of pH 5.0 and 6.00. About 50 fractions with a volume of 5-7 rnl were collected. Two sharp peaks of protein concentration were obtained a t about tubes 5-15 and tubes 27-29. The acid phosphatase activity was localized only in the second peak and represented a 10-fold purification. A substantially different procedure for purification was employed by Ostrowski and his co-workers (03, 0 4 ) . The frozen human prostate gland was sliced into sections and weighed, and 5 volumes of 0.01% Tween 80 solution in distilled water was added. The mixture was homogenized in a Waring Blendor for 30 seconds a t 13,000 rpm, stored in the cold

56

OSCAR BODANSKY

for 24 hr with occasional shaking, filtered through glass wool, and the residue was reextracted with 1 volume of distilled water. The combined filtrates were dialyzed against distilled water for 72 hours, with two changes of water. The pH was adjusted to 7.0 with ammonia, and the extract then centrifuged for 20 minutes a t 3000 rpm. The clear, supernatant, pink solution, designated as FI, was poured off. It contained 2-5 mg protein per milliliter and, upon electrophoresis a t pH 8.4, showed three separate peaks of protein a, b, and c migrating to the anode. Acid phosphatase as determined by hydrolysis of p-nitrophenyl phosphate was found only in fraction b, and diesterase activity, as determined by hydrolysis of bis (p-nitrophenyl) phosphate, was present between peaks b and c and within peak c. The enzyme solution, FI, was brought to pH 7.0; powdered ammonium sulfate was added up to 45% saturation, and the solution was adjusted with ammonia to pH 7.0. The solution was cooled in the refrigerator for 24 hours, then centrifuged for 20 minutes a t 3000 rpm, and the precipitate was discarded. To the supernatant ammonium sulfate was added up to 65% concentration and the pH readjusted to 7.0. After refrigeration as before, the precipitate was collected by centrifugation at 7000 rpm for 20 minutes. The precipitate obtained by treatment with ammonium sulfate between 0.45 and 0.65 saturation was extracted with McIlvaine's buffer solution (0.077 M NazHPOr0.061 M citric acid) of pH 4.0 by stirring a t 5" for 10 minutes. The mixture was then centrifuged at 10,000 rpm for 20 minutes and discarded. The supernatant, containing most of the acid phosphatase and relatively little of the protein of fraction 1 was clarified by centrifuging off sediment at 35,000 rpm for 60 minutes, then dialyzed successively against a large volume of distilled water for 24 hours, and against 0.0175M sodium phosphate buffer of pH 7.0. This preparation, designated as FII, showed on electrophoresis three anodic protein peaks-a, b, c with b as the major peak. The entire acid phosphatase activity was present in the second peak, b; very little diesterase activity was evident, and it was confined to peak c. The enzyme solution was then chromatographed on a DEAE-cellulose column. Elution with phosphate buffer and NaCl gave 5 peaks; the second and third peaks contained the acid phosphatase, with peak two showing a recovery being about 60% of the enzyme originally applied and containing most of the eluted enzyme. Diesterase was present chiefly in the fourth peak. The second peak (fraction FII-2) was rechromatographed on DEAE-cellulose and yielded a symmetrical and high activity peak, indicating a high degree of purification. This was designated a t FIII. Starch gel electrophoresis a t pH 8.5 showed a single sharply

ACID PHOSPHATASEI

57

defined band migrating to the anode; a t pH 4.5 a single sharp band migrating slightly toward the cathode was similarly obtained. This preparation showed a sharp pH optimum a t 4.8 with p-nitrophenyl phosphate as substrate. Although Ostrowski and Tsugita (04) termed this preparation highly purified and homogeneous, they gave no value for the specific activity and hence for its degree of purification from the original prostatic tissue. Davidson and Fishman (D3) submitted a relatively simplified method of purification. Sliced prostate was homogenized for 1 minute in a Waring Blendor with 1 volume of chipped ice and 3-4 volumes of ice-cold Trisscitrate buffer, pH 3.7. The homogenate was filtered in the cold, and the pH of the filtrate was adjusted by adding first an amount of 22% Tris buffer equal to 3% of the volume of the filtrate and enough dilute ammonia solution to achieve a p H of 7.5-8.5. The filtrate was chilled in ice and solid ammonium sulfate added and dissolved to attain 0.65 saturation. The resulting solution was centrifuged in the cold ( O O ) for 10 minutes. The resulting supernatant (No. 1) was discarded, and the precipitate was suspended in a volume of Trisacitrate buffer, pH 3.7, equivalent to no more than 10% of the volume of the original filtrate. Insoluble protein was removed by centrifugation, and the extract was treated with ammonium sulfate to attain 0.45 saturation. This supernatant (No. 2) was now active, but the precipitate could be extracted as before to yield another active supernatant (No. 3). In several preparations, the degree of purification of supernatant solutions 1 and 2, as judged by the specific activity, ranged from about 7- to 40-fold the activity of the original filtrate. 3.2.3. Isoenzymes of Human Prostatic Acid Phosphatase The preceding description of the use of chromatographic methods in the purification of prostatic acid phosphatase (B24, 04) has already indicated that this ensyme exists in more than one molecular form, or isoenzyme. There is, in addition, immunological (S19) and starch gel electrophoretic evidence (L14,L15,524, 531) of the existence of several forms. In order to ensure that no isoenzymes are lost during any purification, it is preferable to perform such studies on a homogenate of the whole tissue. It should be recognized that the isoenzymatic composition may not be characteristic of the prostatic cell per se, but may also represent components from blood cells, secretory ducts, connective tissue, and other sources. Sur et al. (S31) subjected a concentrated aqueous extract of human prostate gland to starch gel electrophoresis in citrate buffer a t pH 6.2, and obtained at least thirteen active zones. These were recovered from

58

OSCAR BODANSKY

the gels in four groups according to their mobilities. All four had the same pH optimum of 5.5 with disodium p-naphthyl phosphate as substrate. In this preliminary report, it was stated that the apparent Michaelis constants, stability a t 47" and p H 6.2, were essentially the same for all four groups. Treatment with butanol to dissociate any possible lypoprotein-phosphatase complex, with an active protease to dissociate any possible complexes with other proteins, or with EDTA to dissociate possible metal-bound complexes failed to alter the electrophoretic pattern. These results were confirmed to a large extent by Lundin and Allison (L14, L15), who examined the electrophoretic patterns of acid phosphatase from different organs and animal forms. We shall concern ourselves only with the results on human tissues. Since there is no statement that equal activities of acid phosphatase from different tissues were placed a t the origin, it is difficult to make any definite conclusions about the patterns from the different tissues. I n general, these tissues showed between 10 and 17 bands upon electrophoresis a t pH 6.0 for a period of about 4 hours. Human prostate had a strong band that moved very little from the origin, and this band was not seen in the other tissues. Smith and Whitby (524) homogenized fresh autopsy specimens of normal human prostate, stripped of capsule, and cut into small pieces in a Waring Blendor a t 4°C for 20 seconds in 4 volumes of 0.01 M citrate buffer (pH 6.0). The supernatant was decanted, filtered a t 4"C, and stored a t -20°C. When a small aliquot, 10 ml, of this homogenate was applied to a column of Sephadex G-200, and the column was eluted upward with 0.01 M citrate buffer (pH 6.0) containing 0.1 M NaCl, two peaks of acid phosphatase activity were obtained. The first peak was small and did not appear if the sample was first centrifuged at 100,OOOg for 30 minutes, and it appeared to be particle-bound enzyme. The second peak contained 90-100% of the applied activity, was always homogeneous, and appeared to consist therefore of enzyme species differing in molecular weight by less than 5%. Calibration of the column indicated a molecular weight of about 105,000. The crude prostatic homogenate was also passed through a column of cellulose phosphate, and eluted with 0.01 M citrate, pH 6.0, The resulting single peak was then fractionated by DEAE-cellulose chromatography into two peaks. These two peaks (fractions I1 and IV) were further purified by gel filtration; they constituted 50- and 100-fold purifications from the prostatic homogenate. There were no marked differences in the relative rates of hydrolysis of a number of phosphate esters a t a concentration of 2 mM by the

ACID PHOSPHATASE

59

two purified fractions. For example, the pattern of hydrolysis on some of these esters by fraction I was: p-nitrophenyl phosphate, 100; naphthyl l-phosphate, 145; naphthyl 2-phosphate, 135; glucose l-phosphate, 2; P-glycerophosphate, 60. The corresponding pattern of hydroIysis of these esters by fraction I1 was 100, 135, 126, 5 , 83. L-( +)-tartrate a t a concentration of 5 mM inhibited the hydrolysis of all esters equally by fraction I and fraction IV. The extent of inhibition was 90-100% for sll the phosphate esters, except for 70-800/0 for the naphthyl phosphate esters. The optimal p H was 4.5 for p-nitrophenyl phosphate and about 6.0 for ,f3-glycerophosphate and adenosine 5'-monophosphate, regardless of whether fraction I1 or fraction I was used. Starch gel electrophoresis of prostatic homogenate was carried out a t 4" in citrate buffer, pH 5.0, 0.5M and 0.1 M in the cathode and anode vessels, respectively, and 5 mM in the gel. An overall potential of 200 V, giving a current of 35 mA was applied for 20 hours. About 20 bands were usually obtained. All bands were almost totally inhibited by 5 mM L - ( +)-tartrate. When a sample of homogenate was digested with neuraminadase for varying periods of time, there was a progressive disappearance of the fastest bands (11-20), until the bulk of the enzyme activity was compressed into bands 3-10, after which these bands were much more slowly digested, and bands 1 and 2 increased in prominence. The results indicated that the enzyme could undergo progressive removal of acidic (probably neuraminic acid) groups. It would seem, therefore, that the electrophoretic heterogeneity of the enzyme arises from a single enzyme protein bearing a variable number of acidic residues. The role of neuraminic acid in the heterogeneity of acid phosphatase from the human prostate gland has been studied more recently by Ostrowski and his associates (05). Slices of frozen prostate were immersed in 3 volumes of 0.01% aqueous Tween 80 solution and left in the cold room overnight with slow mixing, filtered, and squeezed through gauze. The liquid was centrifuged a t 100,OOOg for 60 minutes; the supernatant was dialyzed for 48 hours against distilled water, and then concentrated. This concentrate gave a single peak of acid phosphatase activity during ultracentrifugation in sucrose gradient, during filtration on Sephadex G-100; or on agar-gel suspensions. The enzyme activity was assayed by its action on 0.02 M P-nitrophenyl phosphate. These results indicated that acid phosphatase was a relatively homogeneous protein or else composed of molecules with molecular weights not differing from each other by more than 5 % . Earlier studies with gel filtration (03, S24) had indicated an average molecular weight of about 100,000.

60

OSCAR BODANSKY

As has been noted in the section on the purification of prostatic phosphatase, earlier investigators had observed that chromatography with Sephadex G-200 or with DEAE-cellulose yielded two peaks of enzyme activity (524). Ostrowski et a2. (05) confirmed these findings. A 5-ml sample of a prostatic extract was applied to an equilibrated DEAEcellulose and eluted with phosphate buffer of increasing concentrations and decreasing pH. Fraction I, representing 70-800/0 of the total activity eluted, came off with 0.05M phosphate buffer a t pH 6.5. At about p H 6.0 and a somewhat higher concentration of buffer, fraction 11, containifig the enzyme activity remaining in the column, was eluted. These two fractions were collected, concentrated, and filtered separately on a column with Sephadex G-100. These two fractions were mixed and rechromatographed on a column of DEE,-cellulose. Elution with phosphate buffer of increasing concentration and decreasing pH again resulted in two distinct fractions. These two fractions, designated as enzyme I and enzyme 11, were subjected to isoelectric focusing, and each gave a t least four active peaks. Enzyme I yielded fractions with isoelectric peaks ranging from pH 4.8 to 5.2, and enzyme I1 gave four fractions with peaks ranging from 4.05 to 4.60. When these two acid phosphatases were mixed and digested with neuraminidase and were then submitted to isoelectric focusing, one single symmetrical peak of activity was obtained a t a pH of about 6.15. It was apparent that treatment with neuraminidase abolished the electrophoretic heterogeneity of these two enzymes. The splitting off of neuraminic acid (NANA) produced no appreciable change in the enzymatic activity of either acid phosphatase I or 11. The isolated enzymes were hydrolyzed by trichloroacetic acid and showed liberations of 31 & 2.8 and 40 k 8 moles of NANA per 100,000g of enzyme protein. In the case of enzyme I, the liberation by acid was about 25% higher than that by neuraminidase. These results indicate rather clearly that the large number of isoenzymes of prostatic acid phosphatase which have been demonstrated by gel electrophoresis or isoelectric focusing differ from each other in the number of neuraminic acid residues attached to essentially the same protein molecule. 3.2.4. Kinetics of Human Prostatic Acid Phosphatase Using an approximately 300-fold purified preparation of prostatic acid phosphatases, obtained essentially according to the procedure of London and Hudson (LlO), Tsuboi and Hudson (T3) undertook several types of kinetic studies. These investigators observed that the purified preparation of the prostatic acid phosphatase was highly unstable in

ACID PHOSPHATASE

61

dilute solution and was inactivated rapidly, that a relationship existed between the inactivation process and time of shaking the enzyme preparation and the temperature, and that a surface-active agent like Triton X-100 or various proteins prevented the inactivation. A second important factor governing the reaction velocity was the presence of various ions. The usual Lineweaver-Burk plot of reciprocaI of reaction velocity against the reciprocal of substrate (sodium P-glycerophosphate) yielded a straight line relationship only with dilute substrate concentrations. With increasing substrate concentration, the reaction velocity and consequently the slope increased by an increment in excess of that predicted by theory. Tsuboi and Hudson (T3) explained these effects by assuming that the change in the concentration of substrate, including the cation Na+, constituted changes in the ionic environment and hence accounted for the deviations at the higher levels. With decreasing substrate concentrations, that is, to levels of 0.005 M and below, the ionic differences became negligible in the presence of relatively high buffer concentrations, as, for example, 0.15 M acetate a t pH 5.5. Tsuboi and Hudson (T3) also found that citrate buffer, 0.05 M , or acetate buffer, 0.15 M with 0.01 M EDTA gave substantially higher velocities than acetate alone, and they attributed this effect to the abolition of inhibitory action by contaminant traces of heavy metals. When these factors were taken into account, and velocities were determined at dilute concentrations of substrate and in the presence of 0.1 M acetate, pH 5.5, as buffer and 0.01 M EDTA, it was possible to determine the Michaelis constants for different substrates. At 37°C these values were as follows: a-glycerophosphate, 3.1 mM; p-glycerophosphate, 2.4 mM; yeast adenylate, 0.25 mM; phenyl phosphate, 0.15 mM. The corresponding values for V,,,, the velocity a t infinite substrate concentration, were expressed as micrograms of phosphate liberated per minute: 0.9, 1.0, 1.0, and 1.0. With phenyl phosphate as substrate, L-( +)-tartrate was found to be a strong competitive inhibitor, with M . The enzyme was also reversibly inactivated by K i equal to 0.63 X cupric and ferric ions and by the thiol reagent, p-chlormercuribenzoate. Some years later, Nigam et al. (N3) undertook a kinetic study with preparations that represented an approximately 20- to 30-fold purification (D3). They first presented the time courses of hydrolysis of phenyl phosphate, nitrophenyl phosphate, and P-glycerophosphate, the first two a t an initial concentration of 0.0043 M and the last, /3-glycerophosphate, a t a concentration of 0.0028M. Although the hydrolyses of phenyl phosphate and nitrophenyl phosphate were of zero order for approximately the first 25% of the reaction, that of P-glycerophosphate was of this order for only the first lo%, the reaction velocity decreasing

62

OSCAR BODANSKY

progressively after that. The plot of reaction velocity against enzyme concentration exhibited straight lines for phenyl phosphate and nitrophenyl phosphate and a slightly curved one for P-glycerophosphate. Direct proportionality of a correctly chosen measure of reaction velocity enzyme concentration is a fairly universal characteristic (B20). The p H activity curves were of interest. The optimum pH for hydrolysis of phenyl phosphate wm 4.9, 5.0, and 5.0 in acetate, citrate, and Tris'HC1 buffer solutions, respectively. For nitrophenyl phosphate, the corresponding values were 4.9,4.7, and 5.5,and for P-glycerophosphate, the values were, respectively, 5.5, 5.7,and a range of 5.0 to 6.0. The Michaelis constants, determined a t these optima and in acetate buffer, were 0.75 mM for phenyl phosphate, 0.81 mM for nitrophenyl phosphate, and 4.0 mM for P-glycerophosphate. In the presence of citrate buffer, the corresponding values were 0.091 mM, 0.3 mM, and 2.0 mM. It may be seen that the values for P-glycerophosphate were in fairly good agreement with those obtained by Tsuboi and Hudson (T3), whereas the discrepancies for phenyl phosphate were somewhat greater. With citrate as buffer, the Michaelis constants were 0.091 mM for phenyl phosphate, 0.31 mM for nitrophenyl phosphate, and 2.0 mM for P-glycerophosphate. Of various monocarboxylic and dicarboxylic acids tested a t a concentration of 0.01M , only oxalate, saccharate, and L- (+ ) -tartrate showed substantial inhibition of the action on all three substrates. For example, oxalate inhibited these actions in the standard assay method as follows: phenyl phosphate, 26% ; p-nitrophenyl phosphate, 41% ; P-glycerophosphate, 72%. The comparable inhibitions by saccharate were 42, 64, and 91% and those by L-( +)-tartrate were 96, 97, and 100%. Whereas other carboxylic acids like maleate, glutamate, malonate, and glucuronate did not inhibit the hydrolysis of phenyl phosphate p-nitrophenyl phosphate, the inhibition of the hydrolysis of P-glycerophosphate was fairly substantial, ranging from about 4 0 % . The inhibition by L-( )-tartrate was studied throughout a range of sub&rate concentrations and yielded the following values for the inhibition constant, K i : 0.95 X M with phenyl phosphate as substrate; 4.5 X lod6M with nitrophenyl phosphate and 2.4 X M with a-glycerophosphate. The value with nitrophenyl phosphate was essentially that, 3.4 X M , reported by Kilsheimer and Axelrod (K3). There is no information concerning the isoenzymatic composition of the purified prostatic phosphatases that were used in the preceding kinetic studies. Nor do there appear to be any kinetic studies on the individual isoenzymes. The possibility exists that substantial differences in kinetic characteristics, such as the value for Ki, for L-( )-tartrate,

+

+

ACID PHOSPHATASE

63

reported by different investigators may reflect differences in the isoenzymatic composition of the purified prostatic acid phosphatases which they employed.

3.3. HUMAN ERYTHROCYTIC ACID PHOSPHATASE 3.3.1. Introduction The presence of acid phosphatase in the human erythrocyte was recognized in 1934 (D4) and properties of this enzyme were studied for almost thirty years (A4, K6, T1, T2, T4, T5) before its role in human genetics was revealed (H13). This role will be described in detail later. The properties of crude preparations of erythrocytic acid phosphatase have been previously noted in this review. At this point, we shall describe methods of purification, and the nature of the isoenzymes, particularly as they are related to the phenomenon of polymorphism.

3.3.2. Purification of Human Erythrocytic Acid Phosphatase Tsuboi and Hudson (T2) described a 1500-fold purification. One liter of red cells from outdated blood was thoroughly washed free of leukocytes and plasma proteins with 1% saline, hemolyzed with four volumes of distilled water, and stirred thoroughly with 400 ml of calcium phosphate gel suspension, containing approximately 45 m M tricalcium phosphate per liter. The gel was removed by centrifugation, washed twice with small volumes of distilled water and then discarded. The combined supernatant and washes which contained almost all the original enzyme activity, was now mixed with 1600 ml of calcium phosphate gel suspension. The gel, which had absorbed 90% of the enzyme, was washed by repeated centrifugation until the washings were colorless. The washed gel was resuspended evenly with 1 liter water and mixed for several minutes with 1 liter of 0.3M acetat+O.O3M citrate buffer, pH 4.5. The mixture was then centrifuged, and the supernatant eluate contained 60-70% of the adsorbed enzyme. At this stage, the degree of purification, as compared with crude hemolysate, was between 150and 200-fold on the basis of nitrogen determination. The enzyme solution was then treated with ammonium sulfate to 55% saturation. The precipitated enzyme was centrifuged, dissolved in a minimal volume of water (about one seventy-fifth that of the eluate) and dialyzed overnight against several hundred volumes of 0.01 M acetate, pH 5.0. Any precipitated protein was centrifuged off; the color of the resulting solution was a dark brown red due chiefly to the presence of catalase and hemoglobin. The degree of purification was approximately 400-fold that of the crude hemolysate.

64

OSCAR BODANSKY

The dialyzed enzyme solution was now subjected to a repetition of the preceding procedures : admixture of sufficient calcium phosphate gel to adsorb protein but leave the enzyme in solution; centrifugation and addition of more gel to the supernatant to adsorb the enzyme; elution of the enzyme from the gel with a mixture of 0.15 M acetate and 0.015 M citrate a t p H 4.5; addition of solid ammonium sulfate to the eluate to 55% saturation and precipitation of the enzyme. At this stage, the purifications ranged from 650- to 1100-fold with a recovery of approximately 2&30% of the activity present in the crude red cell hemolysate. Solution of this precipitate, dialysis; treatment with solid ammonium sulfate; and collection of the precipitate appearing between 40 and 55% saturation yielded a preparation that represented a 1500-fold purification. The preparations were stable when left sedimented in the ammonium sulfate sclution. A much purer preparation of acid phosphatase from horse erythrocytes was obtained by Ito et al. (12) by adding the DEAE-chromatography procedure to the method of Tsuboi and Hudson (T2). Since this procedure may be applicable to human erythrocytes, i t will be mentioned briefly. One liter of horse erythrocytes was washed and lysed by the addition of 4 liters of distilled water. One liter of calcium phosphate gel suspension was added to the hemolysate to remove most of the nonenzymatic protein, and the mixture was centrifuged. Five liters of the gel suspension were added to the supernatant, resulting in the adsorption of the enzyme. The enzyme was eluted with citrate-acetate buffer, pH 4.5, and solid ammonium sulfate was added to the eluate up to 60% saturation. The precipitate was collected, dissolved in 40 ml of water, dialyzed against water a t 5°C for 10 hours, and again subjected to calcium phosphate gel adsorption, elution, and precipitation with solid ammonium sulfate to 60% saturation. The precipitate was dissolved in a minimal volume of water (4 ml) and dialyzed against water; the resulting solution was applied to a DEAE-cellulose column which had been equilibrated with 0.01 M sodium pyrophosphate buffer containing 0.08% of the detergent Emargin 810. A linear gradient elution was then carried out with 0.01 to 0.2 M sodium phosphate buffer, pH 6.0. The eluate containing the enzyme was freed of buffer by passage through a Sephadex G-50 column, previously washed with 0.001M mercaptoethanol and 0.08% Emargin 810. The specific activity of the material prior to application on the DEAE column was 15.0, as compared with the specific activity of 0.0139 in the crude hemolysate. This represented a 1080-fold purification, of the same order as that reported by Tsuboi and Hudson (T2). The first part of the enzyme peak coming through the DEAE-cellulose column con-

ACID PHOSPHATASE

65

tained 8.4% of the enzyme originally present in the crude red cell hemolysate and had a specific activity of 120. This represented an approximately 9000-fold purification. 3.3.3. Isoenzymes of Human Erythrocytic Acid Phosphatase In the course of studying chromatography of various proteins on the anion-exchange resin Dowex 2, Boman and Westlund (B25) observed that human erythrocytic acid phosphatase was eluted in two peaks. Several years later, in 1962, Angeletti and Gayle ( A l l ) studied the chromatography of a centrifuged and dialyzed 1:5 human red cell hemolysate on a DEAE-cellulose column. Twenty to 25 ml of the hemolysate was applied to the column; washing with phosphate buffer removed practically all of the hemoglobin. A parabolic gradient salt elution, increasing to a final concentration of 1M NaC1, was started after an effluent volume of 200 ml had been collected. At approximately an effluent volume of 400 ml and an NaCl concentration of about 0.1 M , the acid phosphatase began to emerge. This enzyme consistently appeared in three fairly well separated peaks. Angeletti and Gayle ( A l l ) were able to elicit some differences in the characteristics of these peaks. With p-nitrophenyl phosphate as substrate and citrate as buffer, peak 1 showed optimal activity at pH of about 4.4, whereas peaks 2 and 3 had a p H optimum of about 5.5. Again, the acid phosphatase in peak 1 could be inhibited by 0.02M tartrate to the extent of 40%, whereas peaks 2 and 3 were not inhibited, even up to concentrations of 0.04 M tartrate. Prostatic phosphatase, it will be recalled, is almost completely inhibited by 0.01 M L- ( )-tartrate. Georgatsos (GI) failed to obtain any fractions upon applying whole hemolysates to Sephadex G-75 or G-100. However, when he precipitated the acid phosphatase with acetone, washed the precipitate twice in acetone, then extracted the resulting dry powder with 0.14M NaC1, he obtained an active preparation of acid phosphatase. Application of aliquots of this extract to Sephadex G-75 and elution with 0.14 M NaCl resulted in two peaks. The first peak had two pH optima, one a t pH 5.0 and another a t pH 6.0. It was activated by Mg2+ optimally a t a concentration of 6.6 mM. The second peak had a p H optimum a t 5.0 and was not affected by Mg*+. Conversely, fluoride at a concentration of 10 mM inhibited the enzyme activity in the first peak to the extent of 47% but did not affect that in the second. As Georgatsos (Gl) has pointed out, the conflicting results obtained by different investigators may be due to the change in proportion of these two components as purification proceeds from the crude hemolysate. In 1963, Hopkinson et al. (H13) observed that, when human red cell

+

66

OSCAR BODANSKY

hemolysates were subjected to starch gel electrophoresis, more than one zone of acid phosphatase activity were present. At this time, five patterns, A, BA, B, CA, and CB were detected and were described qualitatively in terms of the relative activity and migration of the zone toward the anode. For example, in type A, the ‘Lfastllzone was generally clearly defined and well separated from the ‘Lintermediate’’zone. I n type BA, the “fast” zone was less sharply defined and appeared to merge with the “intermediate” zone which was very much more intense than in type A. There was also a trace of a “slow” zone. Type B showed no “fast” zone, but had a very strong “intermediate” zone and a fairly intense “slow” zone. These patterns indicated several acid phosphatase variants and implied the existence of a new type of human polymorphism. This subject will be considered in greater detail in Section 5. 3.3.4.

Kinetics and Other Properties of Human Erythrocytic Acid Phosphatase I n 1953, Tsuboi and Hudson (Tl) considered some kinetic characteristics of crude red cell acid phosphatase preparation, similar to that used by Abul-Fad1 and King some years earlier (A3, A4). Unlike the latter investigators, Tsuboi and Hudson (Tl) observed only one pH optimum, around 5.5, with phenyl phosphate, a- and P-glycerophosphates or yeast adenylic (probably a mixture of adenosine 3’- and 2’-monophosphates) as substrates. The enzyme was activated by added magnesium with optimal effect being obtained a t a concentration of 0.01 M . Neither prolonged dialysis nor precipitation by acetone resulted in preparations that showed a greater activation by magnesium. The relative rates of hydrolysis of various substrates by nondialyzed hemolysate a t pH 5.5 and 0.01 M magnesium and expressed as milligrams of phosphorus liberated per hour per milliliter of red blood cells may be illustrated by the following results: phenyl phosphate, 4.57; a-glycerophosphate, 2.74; P-glycerophosphate, 0.51 ; yeast adenylic acid, 0.21. Dialyzed hemolysates did not show any significant difference. I n 1948, Axelrod (A17) and in 1950, Meyerhof and Green (M7) showed that acid phosphatases from various sources were capable of mediating direct transfers of phosphate from suitable donors to suitable acceptors. Tsuboi and Hudson (Tl) investigated this phenomenon by determining the amounts of phenol and phosphorus liberated from phenyl phosphate in the presence of increasing concentrations of an acceptor such as glycerol or methanol. For example, a t 0.0069M phenyl phosphate and 0.69M glycerol, 153 pmoles of phenol and 71 pmoles of phosphate were found to be liberated per hour per milliliter of red blood cells. These results indicated a transfer of 82 pmoles of phosphate to

67

ACID PHOSPHATASE

TABLE 1

COMPARATIVE ACTIONSOF HIGHLY PURIFIED PREPAR.AT1ONS O F HUMAN ERYTHROCYTIC AND PROSTATIC ACIDPHOSPHATASES ON VARIOUS SUBSTRATES~

Relative activities as percent of maximum ~~

Substrate

Prostatic acid phosphatase

100 33 2

100 69

Phenyl phosphate a-Gly cerophosphate 8-Glycerophosphate 3-Phosphoglycerate Glucose 1-phosphate Glucose 6-phosphate Ribose 5-phosphate Adenosine 5'-phosphate Adenosine triphosphate Sodium pyrophosphate Diphenyl phosphate 0

~~~~~

Erythrocytic acid phosphatase

4

1 0 8 3 0 1 0

84

0 19 0 11 60 0 0 0

Based on data of Tsuboi and Hudson (T3, T5).

glycerol. The extent of phosphate transfer increased with the concentration of glycerol. A 1000-fold purified preparation of erythrocytic acid phosphatase was used later by Tsuboi and Hudson (T5) in a reinvestigation of the kinetic properties. The pH activity curve now had a broader maximum centering around pH 6.0. Mg2+no longer had any activating effect a t any p H level. The rate of action varied greatly with the substrate. Table 1 shows the comparative actions of the highly purified preparations of erythrocytic acid phosphatase and a similarly highly purified preparation of prostatic acid phosphatase. The concentration of each substrate was 0.01 M , and all velocities were determined in the absence of magnesium, except for ATP and Na,P,07 which were tested with 0.01 M Mg2+. Reaction velocities were determined a t various concentrations of a-glycerophosphate and of phenyl phosphate a t pH 5.5 and 37" with 0.1 M acetate as buffer and 0.001 M EDTA. A Lineweaver-Burk plot yielded a value of 7 mM for the Michaelis constant with a-glycerophosphate as substrate and 0.9 mM with phenyl phosphate as buffer. It will be recalled that the corresponding values for human prostatic phosphatase were 3.1 mM and 0.15 mM according to Tsuboi and Hudson (T3). Nigam e t al. (N3) had obtained a value of 0.75 mM for phenyl phosphate. In view of the experimental errors inherently involved in the determination of Michaelis constants leading frequently to coefficients

68

OSCAR BODANSKY

of variation of 25-30% (N4), it would appear that no marked distinction can be made between the values for the Michaelis constants for human erythrocytic acid phosphatase and those for the prostatic enzyme. In contrast to the marked inhibition of prostatic phosphatase by fluoride (A4) and L-( + ) -tartrate (A4, F1, K3) , neither the acid phosphatase present in crude hemolysates or in a highly purified preparation from such hemolysates is inhibited by these compounds (A4, T5). Tsuboi and Hudson (T5) also studied the effect of temperature on the hydrolysis of a-glycerophosphate by the erythrocytic enzyme a t the optimum pH, 6.0, for this substrate. Using the Arrhenius plot (A15, B21), namely, log velocity against the reciprocal of the temperature, Tsuboi and Hudson (T5) obtained two straight lines that appeared to intersect a t 26°C and which yielded energies of activation of 13,000 calories a t the lower temperatures up to about 26"C, and 9600 calories for the range from this temperature up to about 50°C. The corresponding values for the energy of activation for prostatic acid phosphatase were 12,000 calories up to 26"C, and 8000 calories above this temperature. The Arrhenius equation has been found to hold, that is, E is constant as T is varied, for a great many chemical reactions (B20), and its reported failure to hold in enzymatic reactions has been shown in many cases to be due to the incorrect use of proper measures of reaction velocity (B20). When correct measures were used, a straight line is obtained between the log reaction velocity and the reciprocal of the absolute temperature in accordance with the Arrhenius equation (B20, B21). It is, therefore, possible that the results reported by Tsuboi and Hudson (T5) reflect the use of improper measures of reaction velocity. An alternate explanation is also possible. Using proper measures of reaction velocity in studying the effect of temperature on the fumarase activity, Massey (M6) obtained a straight line relationship with sodium L-malate as substrate, that is, for the dehydration reaction. I n contrast, with sodium fumarate as substrate, that is, for the hydration reaction, a straight line relationship held only at pH 7. At higher or lower pH levels, the relationship could best be described a t any given pH by two straight lines intersecting a t a critical temperature yielding different energies of activation. At pH levels higher than 7.0, the activation energy a t lower temperatures was lower than that a t higher temperature, whereas a t pH levels lower than 7.0, the activation energy a t the lower temperatures was greater than those a t the higher temperatures. It is, therefore, also possible that the temperature effects described by Tsuboi and Hudson (T5) resemble those by Massey (M6) and may be explained by the formulation of Kistiakowsky and Lumry (K7) that deviations from the Arrhenius relationship are the result of low tem-

ACID PHOSPHATASE

69

perature reversible inhibition by one or more constituents of the reaction mixture. Like many other purified enzymes, a 1000- to 1500-fold purified preparation of erythrocytic acid phosphatase is inactivated, particularly when present in dilute solutions. Investigations by Tsuboi and Hudson (T4) showed that two separate phenomena were responsible for the instability of the purified preparation. First, the addition of very small quantities of synthetic nonionic detergents, like Triton X-100, resulted in a complete stabilization of the enzyme against inactivation due to surface forces. Second, the enzyme was found to be rapidly inactivated by trace amounts of heavy metals which were present or introduced as a contaminant through the use of dialyzing membranes or various reagents. This susceptibility to inactivation suggested the presence of sulfhydryl groups in the enzyme.

3.4. HUMAN LEUKOCYTIC ACID PHOSPHATASE The acid phosphatase activity of leukocytes was studied by Valentine and Beck (B8, V l ) in 1951. There appear, however, to have been no significant attempts to purify the enzyme from this source, or to describe its characteristics. Recently, Szajd and Pajdak (532) indicated the isoenzyme characteristics of leukocyte acid phosphatase, and Li and his associates (L7, L8) studied this problem in greater detail. They suspended a leukocyte preparation, carefully separated from blood, in 5% Triton X-100 to yield a final concentration of 10 X lo6 cells per milliliter and subjected the suspension to six cycles of alternate freezethaw treatment. The suspension was then centrifuged a t lO00g for 15 minutes a t 4"C, and the supernatant was used for electrophoretic studies. Specimens centrifuged a t 100,OOOg for 15 minutes gave the same results. Electrophoresis was carried out a t 4°C for 60 minutes on a 7.5% acrylamide gel matrix containing 0.5% Triton X-100 a t pH 4.0 with a current of 4 mA per tube. The substrate was a-naphthyl phosphate. The values for the normal leukocyte acid phosphatase activity and the normal isoenzyme pattern will be described in connection with the alteration of these in various hematologic and hematopoietic disorders. 3.5. LIVERACID PHOSPHATASE

3.5.1. Introduction Liver acid phosphatase has been of particular interest since the demonstration by de Duve (D7, D8, D9, D10) that acid phosphatase and other hydrolytic enzymes were enclosed in an intracellular structure, the lysosome, of the liver and played an important role in the intra-

70

OSCAR BODANSKY

cellular digestion of foreign and endogenous material. This aspect of acid phosphatase will be considered in detail later in this review. The purification of acid phosphatase from the human liver and the description of its properties do not appear to have been accomplished. Partly, this may be due to the inherent difficulty of obtaining normal, fresh human material in amounts substantial enough for purification. However, because of the cellular and physiological importance of acid phosphatase, i t is advisable to describe in the present section the purifications of the enzyme from rat and bovine liver. Moreover, since these purifications were accomplished with the awareness that acid phosphatase from this source might be present in multiple molecular forms, the descriptions will naturally involve a consideration of the isoenzymes and their properties. 3.5.2. Rat Liver Acid Phosphatase The electrophoretic characteristics of rat liver acid phosphatase were considered by Barka in 1961 (B3). He prepared 10% distilled water homogenates from livers of rat, perfused in situ with cold Ringer’s solution. After freezing at -68°C and thawing five times, the homogenates were centrifuged at 105,500g for 60 minutes and the supernatants, excluding the top lipid-rich layer, were used for electrophoresis on polyacrylamide gels. The soluble acid phosphatases in the supernatant represented 60% of the activity of the total homogenate. Gomori’s acid phosphatase technique with P-glycerophosphate as substrate (G7), the post-incubation coupling azo dye method with sodium 6-benzoyl-2naphthyl phosphate as substrate (RlO), and several other methods were employed in eliciting three bands of acid phosphatase activity. Employing the supernatant obtained by centrifuging a 1:3 rat liver homogenate at 100,OOOg for 60 minutes, Moore and Angeletti (M9) were able to separate by DEAE-cellulose chromatography three major and one minor peaks of acid phosphatase activity. Arsenis and Touster (A16) reported that a partially purified rat liver lysosomal acid phosphatase could be resolved into two enzyme components, a 5’-nucleotidase and a sugar phosphate phosphohydrolase. A 336-fold purification by column chromatography was achieved by Brightwell and Tappel (B32) from lysosomes obtained by differential and density sucrose gradients. The lysosomes were frozen and thawed several times, then centrifuged. The resulting soluble acid phosphatase fraction was dialyzed against suitable buffers and then applied to a DEAE-cellulose column or to a CM-cellulose column. Each column was eluted with a linear &I M NaCl solution, the former a t pH 7.2 and the latter a t pH 5.6. Two peaks of acid phosphatase activity were ob-

ACID PHOSPHATASE

71

tained in each case. The first peak from the DEAE-cellulose column showed a 336-fold purification. The acid phosphatase from the CMcellulose eluate was inhibited by 1.4 mM L-(+)-tartrate to the extent of 80% with p-nitrophenyl phosphate as substrate and to 97% when P-glycerophosphate was the substrate. Purification and crystallization of acid phosphatase from rat liver has been reported by Igarashi and Hollander (11).All procedures were carried out a t 4"C, and all solutions contained 5 mM 2-mercaptoethanol and 1 mM EDTA. Livers from freshly killed rats were immediately homogenized with 50% glycerol in a Waring Blendor for 3 minutes, and the homogenate was centrifuged at 8000 rpm for 20 minutes. About 90% of the activity was recovered in the supernatant; this was adjusted to pH 5.0 with 1 M acetic acid and stirred for 30 minutes. The precipitate that formed was separated by centrifugation and discarded. The supernatant fluid was dialyaed overnight against 5 mM acetate buffer, pH 5.0, and the resulting precipitate was also removed by centrifugation and discarded. The dialyzed material was treated with ammonium sulfate so as to obtain the fraction precipitating between 0.5 and 0.8 saturation. This precipitate, representing a 6.9-fold purification and 53% recovery, was dissolved in a minimum quantity of 0.01M sodium acetate buffer, pH 5.0, and applied to a Sephadex G-75 column equilibrated with 0.01 M sodium acetate buffer. Elution with the same buffer gave a single peak of enzyme activity, but only the aliquots containing higher enzyme activity were pooled and precipitated by addition of ammonium sulfate between 0.5 and 0.8 saturation. The resulting precipitate was dissolved in 5 mM imidazole buffer, p H 7.1, and dialyzed against the same buffer. The enzyme solution was then chromatographed on a DEAE-cellulose column and eluted by a linear gradient composed of a mixture of the buffer and 0.5M NaCl. Two peaks of enzyme activity were obtained, pooled separately and concentrated by precipitation with 0.8 saturated ammonium sulfate. The first peak of enzyme solution was dialyzed against 10 mM sodium succinate buffer, p H 6.0, without EDTA and with 2-mercaptoethanol, then applied to a hydroxyapatite column equilibrated with the same buffer. Stepwise elution with ammonium sulfate in this buffer yielded a single peak in 0.1 M ammonium sulfate fraction. The pooled enzyme was concentrated with 0.8 saturated ammonium sulfate to give 1% protein concentration in imidazole-glycylglycine buffer, pH 7.1. Addition of ammonium sulfate to between 0.5 and 0.55 saturation yielded a turbid solution which in turn yielded crystals after 24 hours a t 4°C. The second peak was applied to a Sephadex G-200 column equilibrated with 0.01 M

72

OSCAR BODANSKY

sodium acetate buffer and eluted with the same buffer as a single peak, termed PI1 enzyme. The properties of these two components or isoenzymes of rat liver phosphatase were similar in many respects, but different in some. Thus, the molecular weight of each was approximately 100,000. With p-nitrophenyl phosphate as substrate, the Michaelis constant was 0.091 ? 0.007 mM for the crystalline isoenzyme and 0.047 5 0.004 for the PI1 component. The isoelectric points of the crystalline and PI1 isoenzymes were pH 7.7 and 4.5, respectively, as determined by the method of isoelectric focusing. The activity of each isoenzyme was completely inhibited by 1 m2M L- ( ) -tartrate or fluoride at a concentration of 1.0 mM p-nitrophenyl phosphate as substrate. The finding by Igarishi and Hollander (11) that rat liver acid phosphatase has two isoenzymes is not in agreement with the observation of Barka (B3), who found three components on polyacrylamide electrophoresis, or of Moore and Angeletti (M9), who were able to separate by DEAE-cellulose chromatography three major peaks and one minor peak of this enzyme activity. Using a lysosomal extract of rat liver Shibko and Tappel (S15) found three fractions by DEAE-cellulose chromatography.

+

3.5.3. Bovine Liver Acid Phosphatase

Heinrikson (H3) has recently submitted a purification and characterization of a low molecular weight acid phosphatase from bovine liver. Five hundred grams of fresh bovine liver, minced in a meat grinder, were extracted at 0 4 ° C with 1500 ml of 0.3 M sodium acetate, pH 5.0, containing 1 mM EDTA (buffer A). The suspension was stirred for 90 minutes at room temperature and then centrifuged a t 4°C for 30 minutes a t 15,OOOg. The supernatant, which amounted to 1400 ml, was stirred with 246 g of solid ammonium sulfate. The mixture was centrifuged for 20 minutes a t 15,OOOg, and the resulting pellet was discarded. The ammonium sulfate concentration of the supernatant solution was increased to 55%. The precipitate, containing most of the enzyme, was centrifuged off, dissolved in 200 ml of 0.3M acetate buffer, pH 5.0, and centrifuged to remove the sediment. The supernatant solution (196 ml) was diluted with 4 volumes of cold 0.1M sodium acetate, pH 5.0; the resulting mixture was chilled to 0" and its pH lowered to 4.15. The suspension was immediately centrifuged and the precipitate discarded. Cold 0.1 M Tris was added to the supernatant to bring the pH back to 5.0, and the solution was centrifuged. The supernatant was again fractionated by the addition of solid ammonium

ACID PHOSPHATASE

73

sulfate, as described previously, so as to obtain the precipitate between

35 and 50% saturation which was centrifuged off and dissolved in 80 mI 0.1 M sodium acetate, 1 m M EDTA, pH 5.0.

The solution was divided into two 40-ml portions, and each portion was added to a column of Sephadex G-75 that had been equilibrated with 0.01 M sodium acetate, 1 mM EDTA, pH 4.8. Elution was continued in the same buffer. Gel filtration of a crude extract of bovine liver on Sephadex-75 had previously given two small peaks and a third large peak of acid phosphatase activity. Elution of the purified 3+50% ammonium sulfate fraction now gave a small peak of about 10% of the enzyme activity, no second peak, and a third peak that contained 90% of the enzyme. The third peak (acid phosphatase 111) represented the low molecular weight component and constituted 30% of the total acid phosphatase present in the 15,OOOg supernatant starting material; the degree of purification was 54-fold. The phosphatase I11 effluent was now chromatographed on a column of sulfoethyl Sephadex C-50. The enzyme was adsorbed, whereas a considerable portion of the nonenzymatic protein passed through ; the enzyme was then washed off with a linear gradient of increasing phosphate concentrations at pH 6.0. The specific activity rose to 360-fold that of the original 15,OOOg supernatant. A small sample was subjected to acrylamide gel electrophoresis and revealed about 1&12 bands. The remainder was concentrated by ultrafiltration under N, to about 30 ml, and solid ammonium sulfate was added to 7576 saturation. The precipitate was separated by centrifugation, dissolved in 1 ml of 0.01 M sodium acetate, pH 5.0, containing 0.1 M NaCl and 1 mM EDTA. Ammonium sulfate was removed by gel filtration on a column of Sephadex G-25. The enzyme solution was added to a column of sulfoethyl Sephadex C-50 and eluted with a linear gradient of increasing NaCl concentration and an increasing, nonlinear gradient of pH. The phosphatase emerged well behind the bulk of the eluted protein; the protein and activity curves of the enzyme peak were coincident. The specific activity was 4300-fold that of the original 15,OOOg supernatant. Acrylamide electrophoresis revealed a single band. The liver acid phosphatase 111 thus isolated had a molecular weight of 14,000 daltons as determined by filtration through a column of Sephadex G-75 that had been calibrated with markers of known molecular weight, and a molecular weight of 16,500 dalt,ons on the basis of sedimentation equilibrium analysis. With p-nitrophenyl phosphate as substrate, the pH optimum was 5.5 and the Michaelis constant was 0.75 mM. The stability of the enzyme at 25" was dependent on pH and

74

OSCAR BODANSICY

the nature of the buffer. The presence of Mgz+or mercaptoethanol in the incubation mixtures led to rapid inactivation of the enzyme, whereas EDTA exhibited a stabilizing effect. The substrate specificity of liver acid phosphatase 111 was of particular interest. With the activity with p-nitrophenyl phosphate set a t 100 and under standardized conditions, the relative activities with the following substrates were : flavin mononucleotide, 68 ; galactose 6-phosphate1 39; glucose l-phosphate, 14. The relative activities with other hexose or ribose phosphates were 1 to 2, and those with a-glycerophosphate and P-glycerophosphate were 3 and 0, respectively. It will be recalled that for prostatic phosphatase, the ratio of the activity with p-glycerophosphate as substrate to that with p-nitrophenyl phosphate was considerably higher-about 60%. These relative activities of different tissue phosphatases are of importance in understanding the sources of serum acid phosphatase activities in various diseases. 3.6. SPLEENACIDPHOSPHATASE In 1957, Singer and Fruton (S22) obtained from beef spleen a preparation that represented a 50-fold purification of phosphoprotein phosphatase, phosphoamidase, and phenylphosphatase, as measured by the hydrolysis of the corresponding appropriate substrates a t p H 6.0. This type of preparation was purified further by Glomset (G5)by dissolving 1 g of the enzyme preparation in 4 ml of distilled water, filtering through a column containing 20 g of TEAE-cellulose in equilibrium with 0.01 M TrisSHCI, and displacing it with the same buffer solution. The enzyme, phosphoprotein phosphatase, emerged in the first protein peak; the succeeding peaks contained little activity. This step represented a 4.8-fold purification. The enzyme material was then subjected to three consecutive electrophoresis a t pH 5.6 on “Pevikon,” a polyvinyl acetate-polyvinyl chloride supporting medium. The third electrophoresis showed a single protein peak which contained the enzyme, and represented a 17-fold purification of the Singer and Fruton (S22) preparation. Although this procedure was designed to purify phosphoprotein phosphatase activity, the final preparation also showed phosphoamidase and phosphatase activity a t pH 6.0 as determined on p-nitrophenyl phosphate as substrate. Its action on glycerophosphate or bis (p-nitrophenyl) phosphate was negligible. In 1966, Chersi e t al. ( C l ) submitted a procedure for isolating a highly purified preparation of acid phosphatase from hog spleen. The starting material was a crude spleen nuclease I1 which contained 110-120 units of acid phosphatase per kilogram of ground spleen and had a specific activity of 0.2-0.3. Chromatography on DEAE-Sephadex A-50 yielded

ACID PHOSPHATASE

75

two major protein peaks of which the first contained most of the acid phosphatase. The specific activity had now risen to 1.65. This fraction was chromatographed on hydroxyapatite; the eluate consisted of four major protein peaks of which the second contained phosphodiesterase and the third, acid phosphatase. The specific activity had risen to 7.05. The third step consisted in loading the acid phosphatase peak on a Sephadex G-100 column equilibrated and then eluted with 0.1 M acetate buffer, pH 5.6. The acid phosphatase came off before the main protein peak and had a specific activity of 78.4. For the fourth and final step, the active fraction was applied to a CM-Sephadex C-50 column equilibrated with 0.1 M acetate buffer, pH 5.6, and was eluted by a gradient, 0.1 to 0.3 M , of acetate buffer a t a molarity of about 0.26 M . It was again applied to the same column and eluted at 0.26 M acetate buffer. This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease 11. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows : p-nitrophenyl phosphate, 100; 5’-AMP, 63; P-glycerophosphate, 60; ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 mM. Phosand pH 4.8. The Michaelis constant at 37°C was 7.25 X phate and chloride ions acted as competitive inhibitors. 3.7. HUMANPLACENTAL ACIDPHOSPHATASE I n 1959 Ahmed and King (A5) determined the properties and activities of placental acid phosphatase. The tissue was washed free of blood by perfusion with 0.9% NaCl, blotted, minced, and homogenized with a n equal volume of water in a Waring Blendor for 2 minutes and centrifuged to obtain the supernatant solution. (The time and speed of centrifugation were not given.) The activity was expressed as milligrams of phenol liberated in 1 hour from phenyl phosphate at p H 4.9. The mean activity in a series of 10 placental extracts was 2.4 units per gram of wet tissue. Since the addition of formaldehyde in the assay system inhibited the activity to the extent of from 0 to 100% in the individual placental extracts and, on the average, of about 50%, it was concluded that the placental acid phosphatase consisted of two components. More recently, DiPietro and Zengerle (D13) studied the properties of acid phosphatase obtained from homogenates of perfused placentas centrifuged a t 6OOg for 5 minutes to eliminate cellular debris. The resultant supernatant was then centrifuged at 96,6009 for 45 minutes in

76

OSCAR BODANSKY

the Spinco No. 50 rotor. This high speed supernatant, which contained about 40% of the total acid phosphatase, was chromatographed on Sephadex G-200.Three peaks of acid phosphatase activity, designated as phosphatases I, 11, and 111, were obtained. The molecular weights were estimated by sucrose density gradient centrifugation and were, respectively: >200,000;105,000,and 35,000. When the homogenate was centrifuged a t 20,0009 (time not given) and the pellet washed with 0.25 M sucrose, resedimented and rehomogenized in 0.25M sucrose containing 1% (w/v) of Triton X-100, the supernatant resulting from centrifugation a t 100,OOOg contained about half of the acid phosphatase bound to the particles. As will be seen presently, this phosphatase, designated as isoenzyme P for convenience, resembled acid phosphatase I1 in several respects. DiPietro and Zengerle (D13) did not describe the degree of purification of these three isoenzymes with respect to the original homogenate or, indeed, pursue their further purification. However, the properties of these isoenzymes were investigated in considerable detail. Isoenzymes I and I11 had pH optima near 5.5,whereas isoenzyme I1 had a p H optimum in the vicinity of p H 4 and resembled isoenzyme P in this respect. Incubation in 0.05M sodium citrate, pH 4.9,for 15 minutes a t various temperatures showed complete thermal inactivation of isoenzyme I11 a t 55"C, whereas isoenzyme I was inactivated only to the extent of 45%) and isoenzymes I1 and P to the extent of 10-200/0. The thermal inactivation of this latter pair generally followed an S-shaped curve, with about 50% inactivation occurring a t about 60",and complete inactivation a t 65-70".The inactivation of isoenzyme I was more gradual. The Michaelis constant, K,, with p-nitrophenyl phosphate as substrate was 1 mM for isoenzyme I11 and 7 mM for isoenzyme 11. Table 2 shows the extent of inhibition effected by various substances on the activity of the three isoenzymes. Assays were carried out in 0.05M sodium citrate, pH 4.9,a t 37°C. The concentration of substrate was 0.0055M p-nitrophenyl phosphate; the reaction was allowed to proceed for 15 minutes, then was stopped by the addition of sodium hydroxide. Units were expressed as micromoles of substrate hydrolyzed per minute per milliliter of enzyme solution. It may be seen that p-choromercuribenzoate completely inhibited isoenzyme 111, while affecting isoenzymes I and IT only slightly. The inhibitions by L-( +)-tartrate and fluoride were in the reverse directions. The pattern of hydrolysis of various substrates by these three isoenzymes also showed marked differences. When the velocity of hydrolysis of p-nitrophenyl phosphate was arbitrarily set a t 100,the rates for isoenzyme I were a-naphthyl phosphate, 59 ; pyridoxine 5-phosphate1 40.

77

ACID PHOSPHATASE

TABLE 2 OF ISOENZYMES OF HUMAN PLACENTAL PHOSPHATASE~ INHIBITION Inhibition of Concentration Compound p-Chloromercuribenzoate I.-(+)-Tartrate Fluoride Pyridoxine Pyridoxine 5-phosphate a

Isoenzyme Isoenryme Isoenryme I I1 I11

(mM)

(%I

0.001

14 41 51

20 50

50 10

11

9

(%I

(%I

7

100

90 23 35 27

5 58

0

67

Based on data of DiPietro and Zengerle (D13).

The corresponding rates for isoenzyme I1 were 50 and 66, and those for isoenzyme I11 were 7 and <2. The velocity of hydrolysis of other phosphate esters, such as glucose 6-phosphate or glucose l-phosphate were, in general, low or negligible. An interesting property of isoenzyme 111, not shared by either I or 11, was the stimulation of its action on p-nitrophenyl phosphate by various purines. For example, 5 mM adenine increased the activity by 83%, and 0.1 m.M N5-benzyladenine or Nsmethyladenine by 35% and 3476, respectively. 6-Ethylmercaptopurine at a concentration of 1 mM had a stimulatory effect of 168%. 4.

lntracellular Distribution of Acid Phosphatare

4.1. INTRODUCTION Since normal human tissues are with rare exceptions (R3) seldom available in amounts adequate for study of intracellular distribution of acid phosphatase or other enzymes, most of our information in this area must be based on investigations in animals. I n 1951, while engaged in studying the specific glucose-6-phosphatase of rat liver, Berthet and de Duve (B14) noted th at a large proportion of the acid phosphatase in this tissue was apparently linked with the mitochondria. However, further investigations (A12, A13) showed that this binding involved, not the mitochondria as such, but rather cytoplasmic granules which were recovered mainly with the mitochondria and, to some extent also, with microsomes. Indeed, the acid phosphatase appeared to be present in a specific LLsaclike” structure, the permeability and integrity of which could be altered. Decreasing the concentration of sucrose which had been used in the centrifugal separation of the fraction containing acid phosphatase or even homogenizing this fraction in distilled water resulted in consider-

78

OSCAR BODANSKY

able increases of the velocity of action of the enzyme on the substrate, sodium P-glycerophosphate. The acid phosphatase appeared to be associated in the saclike structure with other hydrolytic enzymes, such as P-glucuronidase and cathepsin, which also acted optimally a t acid pH levels. Further studies were undertaken to isolate this structure (A13, G2). By means of a differential centrifugation procedure which will be described in detail later, de Duve and his associates (D9, D10) determined the intracellular distribution of total and free acid phosphatase activity and of other enzymes as well. The mean values, expressed as percent of total acid phosphatase activity, were: nuclear, 3.6; mitochondrial, 24.1 ; light mitochondrial, 40.7; microsomal, 20.1 ; final supernatant, 13.3. Several other rat liver acid hydrolases, such as ribonuclease, deoxyribonuclease, cathepsin, and P-glucuronidase, showed a similar intracellular distribution, with a preponderance of activity in the light mitochondrial fraction, in the light and heavy mitochondrial fractions, or in the light mitochondrial and microsomal fractions. The similar pattern of distribution, of these enzymes and acid phosphatase, led de Duve and his associates to the provisional conclusion that these belonged to granules of the same class. For practical purposes, it was proposed to refer to these granules as lysosomes, thus calling attention to their richness in hydrolytic enzymes (D10). Using electron microscopy, Novikoff et al. (N6) found that rat liver fractions rich in these “1ysosornalJ1enzymes, particularly acid phosphatase, showed the presence of mitochondria but had a predominance of single-membrane-limited bodies which were generally electron dense. Fractions with a low acid phosphatase activity rarely showed dense bodies. This observation provided some correlation between the biochemical concept of lLlysosomes”and the existence of a structural unit within the cell (528). The question arises whether acid phosphatase is present only in lysosomes or whether it exists also in other organelles of the cell (B4). We have just noted that centrifugal methods used by de Duve and his associates (D10) showed a preponderance in, but not a complete confinement of, the acid phosphatase to one ultracentrifugal fraction. The Gomori method (G7) for the definition of acid phosphatase activity, particularly when combined with electron microscopy (N5), has also failed to show exclusive localization in one organelle. It is possible that such staining may be, and indeed has been, regarded as an enzyme-dependent artifact, resulting from a diffusion of reaction intermediates, reaction product, or enzyme ( D l ) .

ACID PHOSPHATASE

79

4.2. INTRACELLULAR DISTRIBUTION OF ACID PHOSPHATASE IN LIVER Of several methods that are potentially available for determining the intracellular distribution of acid phosphatase and other enzymes, the chief ones currently in use are ultracentrifugal separation and histochemical examination. Each of these has its disadvantages and advantages, some of which have already been indicated. At this point we will consider the quantitative ultracentrifugal methods. After considerable preliminary work, de Duve e t al. (D10) proposed the following procedure for rat liver acid phosphatase. Albino adult rats, fasted for 12 hours, were killed by a blow on the head and bled. The liver was quickly taken out, immersed in an ice cold medium, weighed, cut, and dispersed with 3 volumes of 0.25 M sucrose in a homogenizer of the Potter-Elvehjem type. After a single run upward against the rapidly rotating pestle, the resulting slurry was centrifuged in the cold a t 10,OOOgmin. The sediment, which contained nuclei and unbroken cells, was rehomogenized and centrifuged by 6000g-min twice, and the final sediment made up to a volume equal to 4 times the weight of tissue processed, yielding the 1:4 nuclear fraction. The supernatants were combined and made up to volume to form the 1 : l O cytoplasmic extract, which was further fractionated. Three particulate fractions were successively isolated by integrated forces of 33,000g-min, 250,000g-min and 3,000,000~-min.The washed granules were finally taken up in small volumes of 0.25 M sucrose. The “free” acid phosphatase activity was determined on the various fractions by incubation for 10 minutes a t 37°C in the presence of the substrate, /3-glycerophosphatase, and of sufficient sucrose to make the concentration of this sugar 0.25M in the mixture. Total acid phosphatase activity was measured in a similar manner on preparations, usually diluted 10-fold in distilled water and exposed for 3 minutes to the action of the Waring Blendor. Care was taken to avoid overheating by the blender, and sucrose was added to a 0.25M concentration in the final assay mixture. The distribution of total and free acid phosphatase activity in various subcellular fractions was determined in 19 experiments, and the mean values for the total acid phosphatase in the fractions, expressed as percent of the total acid phosphatase activity in the cell, were: nuclear, 3.6; heavy mitochondrial, 24.1; light mitochondrial, 40.7 ; microsomal, 20.1 ; final supernatant, 13.3. As may be seen, the light mitochondrial fraction, as obtained by de Duve et al. (DlO), contained only 40.7% of the acid phosphatase of the rat liver. Starting with this fraction, Sawant e t al. (54) attempted

80

OSCAR BODANSKY

to isolate the lysosomal entity and determine its properties. Male Sprague-Dawley rats were sacrificed by decapitation, and the livers were washed free of blood with cold 0.25 M sucrose. All subsequent operations were performed at 0-4".The livers were homogenized in 0.25M sucrose (1:8, w/v) for 30 seconds in a Waring Blendor a t top speed. The pH was adjusted to 7.2 with 5 N KOH, and the homogenate was then filtered through muslin. The homogenate was centrifuged at 7509 for 10 minutes; the pellet was discarded, and the supernatant was then centrifuged a t 3009 for 10 minutes, The resulting pellet contained "heavy" mitochondria and was discarded, whereas the supernatant was then centrifuged at 16,3009 for 20 minutes. The supernatant was now discarded, and the resulting pellet which contained what de Duve and his associates had originally termed light mitochondria (D10) was designated as fraction FI. This fraction, FI, as well as fractions FII and FIII, were collected by means of a GSA rotor ( r = 16.3 cm) which had compartments for 250-ml bottles. Acid phosphatase in fraction I had 4.2-fold the specific activity of the enzyme in the original homogenate. The degree of purification of other lysosomal enzymes, namely, aryl sulfatase, ribonuclease, and cathepsin were of the same order of magnitude. The pellet representing FI was resuspended in 0.3M sucrose and centrifuged a t 9500g for 10 minutes. The supernatant was discarded, and the pellet, designated as FII, was essentially the light mitochondria1 fraction. It was resuspended in 0.45 M sucrose, layered over a discontinuous gradient of 0.7M sucrose (bottom layer) and 0.6M sucrose (middle layer) and centrifuged a t 95009 for 30 min. The supernatant was discarded, and the pellet (FIII) was resuspended in 0.7 M sucrose. At this point, the specific activities of lysosomal enzymes in the suspended pellet with respect to the specific activities in the original homogenate as 1 were as follows: acid phosphatase, 23.0; aryl sulfatase, 55; ribonuclease, 20.5; cathepsin, 18. The suspension was centrifuged at 59009 for 30 minutes. The pellet was discarded, and the supernatant was carefully decanted. This was centrifuged a t 17,OOOg for 20 minutes. The resulting pellet was again washed with 0.7 M sucrose, resuspended, and centrifuged with a SS-34 rotor ( r = 10.6 cm) in 50 ml tubes to yield the final fraction, FIV. The acid phosphatase activity of this pellet was 5499 nmolesJminute per milligram of N as compared with an activity of 82 nmoles/minutes per milligram of N for the original homogenate. This represented a 67-fold purification; the degrees of purification for other lysosomal enzymes were: aryl sulfatase, 200; ribonuclease, 72; cathepsin, 62. The measurement of the activities of succinoxidase, uricase, and glucose-6-phosphatase as representing the presence of mitochondria,

ACID PHOSPHATASE

81

peroxisomes, or microsomes, respectively, indicated the absence of the first two types of particles in fraction I V and only a 6 7 % contamination by microsomes. The yield of acid phosphatase was 11%, as compared with 30% for aryl sulfatase and 15% for ribonuclease. Obviously, in the attempt to obtain pure preparations of intracellular components, it is inevitable that losses be encountered. Such procedures are therefore not suited for obtaining an estimate of the quantitative distribution of intracellular components. Approximately 5-974 of the lysosomal enzymes-aryl sulfatase, acid phosphatase, and ribonuclease-were present in the free form. The remaining 91-95% of the activities of these enzymes were in the latent form and required alteration of the permeability or disruption of the lysosomal membrane to become active. It has been estimated that approximately ?MOP of the acid phosphatase in rat liver can be recovered in the lysosomal fraction, the remainder being distributed between the soluble fraction and other subcellular fractions (D9, S15). The question therefore arises concerning the extent to which this remainder is derived from lysosomes broken during the fractionation procedure or whether some acid phosphatase is actually localized in subcellular structures, other than lysosomes. Shibko and Tappel (S15) approached this problem by comparing the properties of the acid phosphatase in the lysosomal fraction isolated in the manner just described with the properties in the mitochondrial, microsomal, and soluble fractions. Of eleven substrates tested, only F-1,6-diP, AMP, and p-nitrophenyl phosphate were hydrolyzed a t rates approaching that, or greater than that, of ,f3-glycerophosphate. Some of these are shown in Table 3. The microsomal fraction hydrolyzed glucose 6-phosphate very readily, such hydrolysis obviously reflecting the presence of glucose-6-phosphatase, the characteristic enzyme of this fraction. Although the activities in the soluble fraction were about 1-276 of those in the lysosomal fraction, the relative actions on the various substrates were essentially the same. The lysosomal and the soluble fractions of acid phosphatase were compared in several other ways. The K , values (mM) for the lysosomal fractions on various substrates were: P-glycerophosphate, 1.6 ; fructose l16-diphosphate, 2.0; p-nitrophenyl phosphate, 1.6; AMP, 0.43; they were not significantly different from those obtained with the soluble fraction. The pH-activity curves with these substrates were similar for the two fractions. Inhibition of phosphatase activity of the lysosomal and soluble fractions occurred a t approximately the same concentration of fluoride or L- ( )-tartrate when P-glycerophosphate, AMP, or fructose 1,6-diphosphate were used as substrates. However, with p-nitrophenyl phosphate

+

82

OSCAR BODANSKY

TABLE 3 SUBSTRATE SPECIFICITY OF ACIDPHOSPHATASE IN VARIOUS SUBCELLULAR OF RAT L I V E R ~ . ~ FRACTIONS ~~

Fraction -~~ ~

Substrate -~

Lysosomal

Microsomal

Soluble

4550 6000 640 3860

425 520 4100 840

46 95 10 29

~

8-Glycerophosphate p-Nitrophenyl phosphate Glucose 6-phosphate Fructose 1,g-diphosphate a

Mitochondrial 61

78

51 64

Based on data of Shibko and Tappel (515).

* The activities, expressed as nmolw/mg N/min,

were determined from the hydrolysis of 0.05 M substrate in 0.1 M acetate buffer (pH 5.4) plus suitably diluted subcellular fraction in a total volume of 1 ml during a 15-minute period at 37".

as substrate, the acid phosphatase was relatively insensitive. For example, a t 0.02 M inhibitor, lysosomal acid phosphataee was inhibited to the extent of about 95% by L - ( +)-tartrate and about 80% by fluoride; the inhibitions of the soluble acid phosphatase were about 25% and 40%) respectively. The electrophoretic patterns of the soluble fraction and of a mixture of the soluble and lysosomal fractions were essentially the same. The acid phosphatase was located in two regions: a distinct band migrating toward the anode, and a less distinct area that moved toward the anode. Chromatography on DEAE-cellulose yielded 3 peaks for each fraction. Relatively larger amounts of fluoride and L- ( ) -tartrate-insensitive acid phosphatase were present in the soluble fraction. That the properties of lysosomal acid phosphatase do not change upon solubilization was evident when the lyeosomal fraction was subjected to alternate freezing and thawing for 10 times and then centrifuged a t 100,OOOg for 1 hour; 50% of its acid phosphatase was released into the suspending medium, and the remainder was associated with the lysosomal membrane and precipitable. The patterns of hydrolysis of various substrates with the exception of G-6-P which reflected membrane-bound glucose-6-phosphatase, were the same for the two subfractions. These results indicate that the major portion of the soluble acid phosphatase is similar to that of the lysosomal phosphatase and may be derived from the injury or breaking of these particles. On the other hand, the presence of a phosphoprotein phosphatase in the soluble fraction and its absence from the lysosomes, and the existence also of a fluoride and an L- ( ) -tartrate-insensitive acid phosphatase in the soluble fraction

+

+

ACID PHOSPHATASE

83

indicate intracellular sources of acid phosphatase, other than lysosomes. When histochemical techniques for acid phosphatase were carried out at the electron microscopic level (N5, N6), dense bodies about 0 . 4 , ~in diameter having a single outer membrane were observed in intact cells of the liver. I n addition bodies with similar morphological characteristics were found to bc located along the fine bile canaliculi. Indeed lysosomes as a whole showed considerable polymorphism, apparently the result of their association with the different materials that are phagocytized by the cell (S29). 4.3. INTRACELLULAR DISTRIBUTION OF ACID PHOSPHATASE IN OTHERTISSUES 4.3.1. Introduction

Most of our knowledge of lysosomes arises from studies of these particles in rat liver. These studies have also supplied considerable evidence that, by virtue of its more than a dozen hydrolytic enzymes, the lysosome can play a role in digesting material foreign to the cell, its own cell, or that its enzymes may be discharged outside the cell to produce lytic effects. It is also possible, as has been shown for rat liver ( R l ) , that lysosomes may be heterogeneous in terms of their enzyme contents. We shall now examine the extent to which acid phosphatase is distributed intracellularly in tissues other than the liver and in species other than the rat. An approximate idea of such a distribution is available from the study of Shibko et al. (S17). These investigators made a 20% homogenate of washed tissue in 0.25M sucrose containing 1 m M EDTA. This homogenate was divided into two fractions as follows. A preliminary centrifugation a t 7509 for 10 minutes (7500g-min) removed unbroken cells and nuclei. The supernatant solution was filtered through glass fiber and recentrifuged a t 75,0009 for 45 minutes (3,375,0009-min) . The resulting pellet was suspended in 0.25M sucrose; on the basis of considerations that have already been presented, this suspension would consist of mitochondria, lysosomes, and most of the microsomes. Shibko et al. (S17) analyzed it for various hydrolytic enzymes, including acid phosphatase. Obviously, designating these enzymes as “lysosomal” is based on the assumption that negligible amounts of these enzymes are present in the other organelles, such as mitochondria and microsomes, that are centrifuged down between 75009-min and 3,375,000g-min. With this understanding in mind, the values of Shibko et al. (S17) may be presented. For livers of various species the total acid phosphatase activity, expressed as nanomoles of substrate hydrolyzed per milligram

84

OSCAR BODANSKY

of N per minute, were: rat, 455; sheep, 26; hog, 15; ox, 12. The fraction, centrifuged down by 7509 for 10 minutes, had the following acid phosphatase activities: sheep, 39; hog, 22; ox 20. These values would apparently constitute a substantial portion of the acid phosphatase in some of the species, but it is difficult to conclude from the study by Shibko et al. (S17) that these represent acid phosphatase in the nuclei or in the unbroken cells. For the spleens of various tissues, the 7500g-min and 3,375,000g-min fractions had the following activities, respectively, again expressed as nanomoles per milligram of N per minute: ox, 116 and 54; hog, 162 and 0 ; sheep, 50 and 10. It would, therefore, appear that in spite of unbroken cells being centrifuged down a t 7500g-min a substantial portion of the acid phosphatase would appear to reside in or attach to the splenic nuclei. Histochemical techniques have indicated that acid phosphatase need not be confined to the lysosomes. For example, with P-glycerophosphate as substrate, this enzyme has been found to be present in the small vesicles in the Golgi region, in vesicles without apparent relationship to the Golgi region and in other elements resembling cisternae of smooth endoplasmic reticulum of cells of rat kidney adenomas (S14), and in the Golgi region of the rat anterior pituitary (525). Kalina and Bubis ( K l ) observed that the acid phosphatase activity in small neurons of rat spinal cord appeared as granules, i.e., lysosomes, whereas the activity in large neurons was localized both in scattered granules and in a network of filaments, representing rough endoplasmic reticulum. Fixation in glutaraldehyde for 30 minutes or in formaldehyde for 4 hours abolished the acid phosphatase activity in the large neurons, but did not affect that in the small cells. Inhibitors like sodium fluoride also showed differences between the large and small cells, and indicated the possible existence of two types of acid phosphatase acting on P-glycerophosphate. Maggi ( M l ) has reviewed other instances in which acid phosphatase acting on P-glycerophosphate is located in intracellular components other than lysosomes. 4.3.2. R a t Heart Employing as a criterion for lysosomal localization the activity of hydrolases in the fraction centrifuged down between 7509 for 10 minutes and 75,OOOg for 45 minutes and treated with the detergent X-100, Shibko e t al. (S17) found no evidence of acid phosphatase in the lysosomal moiety or indeed in the homogenate as a whole of pigeon heart muscle. Romeo e t al. (R8)subjected a mitochondria1 suspension of beef heart tissue to isopycnic centrifugation in sucrose-water solutions and isolated a particulate fraction ( d = 1.174) that showed a high concentration of

ACID PHOSPHATASE

85

latent hydrolytic enzymes and a comparatively low concentration of cytochrome c oxidase and appeared to have the main features of a lysosomal fraction. /3-Galactosidase, p-glucuronidase, cathepsin, acid ribonuclease, and acid deoxyribonuclease were present. No mention was made, however, of the presence of acid phosphatase. Maggi (M2) undertook to reexamine the relationship between the number of lysosomes and the degree of acid phosphatase activity in the rat heart. After homogenization of the minced heart tissue in 0.28 M sucroseEDTA, pH 7.2, the homogenate was centrifuged a t 2400 rpm for 10 minutes to get rid of unbroken cells, nuclei, and some heavy mitochondria. The supernatant was then centrifuged a t 20,000 rpm for 30 minutes in a Spinco Model L. The pellet was resuspended in sucrose-EDTA and frozen and thawed six times before acid phosphatase activity was assayed. With p-nitrophenyl phosphate as substrate, the activity of the supernatant was 2243 nmoles of p-nitrophenol liberated per hour per milligram protein and was four times the activity of the pellet. The pH optimum lay between 5.6 and 5.8. This acid phosphatase activity was not affected appreciably by the chlorides of Mg2+,Ca2+,Na+, K', and only partially inhibited by up to 100 mM sodium fluoride. When p-glycerophosphate was used as substrate and the pellet treated in the same manner, no enzyme activity could be detected in the supernatant, whereas the activity of the pellet was 77 nmoles of P liberated per hour per milligram of protein. This activity was greatly stimulated by 10 mM Mgz' and was inhibited completely by 10 m M sodium fluoride. The pH optimum lay between 4.8 and 5.0. These results would indicate that rat heart contains more than one acid phosphatase, possibly in different subcellular compartments. 4.3.3. Pancreas (Mouse) The distribution of acid phosphatase in the mouse pancreas cell was studied by Van Lancker and Holtzer (V2) by means of centrifugal methods. I n general, the pancreas was homogenized a t 0" for 3 minutes in 0.25 M sucrose with the Potter-Elvehjem homogenizer equipped with a Teflon pestle. The homogenate was diluted to a concentration of 1 g of tissue in 5 ml of total suspension. The suspension was then subjected to successive centrifugations: each pellet was homogenized and washed once and the washing was added to the supernatant. Triton X-100 was added to the assay mixture to assure complete liberation of acid phosphatase. The distribution as percent of the total was as follows: nuclei (6 X 103g-min) 5.0; zymogen granules (8.4 X 103g-min) 5.7; large and small mitochondria (16.8 X lo3 to 263 X 103g-min) 29.8; microsomes (1585 X lo3 to 3170 x 103g-min) 19.8; postmicrosomes (6340 X 103gmin) 7.1 ; supernatant, 19.2.

86

OSCAR BODANSKY

The centrifugal method of separation employed by Van Lancker and Holtzer (V2) was among the earlier ones in the field, and there was probably considerable cross contamination of the fractions. Nonetheless, the distribution seems more disperse than that obtained by de Duve et al. (D10) for rat liver with a comparable method. For example, in the case of the mouse pancreas the small mitochondrial fractions, c, d, and e, obtained by centrifugation between 17 X lo3 and 263 X 103g-min contained 27% of the acid phosphatase and the succeeding ‘(microsomal” fractions, f and g, obtained by centrifugations between 263 X 103g-min and 3170 X 103g-min, contained 24% of the acid phosphatase (V2). For rat liver, comparable fractions, obtained by centrifugation between 33 X los to 250 X 103g-min and 250 X lo3 to 3000 X 103g-min contained 41 and 2076, respectively (D10). 4.3.4. Kidney ( R a t )

Roche and Baudoin (R7) first noted that the kidney contained acid phosphatase. Employing homogenates of rat kidney in 0.45 M sucrose and centrifugations in the same concentration of sucrose, Shibko and Tappel (S16) defined four subcellular fractions as follows: unbroken cells and nuclei, 2.5 minutes a t 6509; lysosomal-mitochondria1 fraction, 1 minute a t 10,OOOg; mitochondrial-microsomal fraction, 20 minutes a t 12,0008; microsomal fraction, 60 minutes a t 10,OOOg; soluble fraction, that remaining as supernatant from the last centrifugation. The enzyme activities of the subcellular fractions were measured after freezing and thawing 10 times. With sodium P-glycerophosphate as substrate, the activities of acid phosphatase expressed as nanomoles of substrate hydrolyzed per minute per milligram of protein, were: homogenate, 53 ; lysosomal fraction, 847 ; mitochondrial fraction, 212, microsomal, 74 ; soluble fraction, 16. With p-nitrophenyl phosphate as substrate, the activities, again expressed as nanomoles of substrate hydrolyzed per minute per milligram of protein, were 52, 507, 146, 82, and 40, respectively. It may thus be seen that the lysosomal fraction defined by Shibko and Tappel (S16) as that centrifuging down in 0.45 M sucrose for 1 minute a t 10,OOOg was the most active. However, a more active fraction could be isolated by methods similar to that employed by Sawant et al. (S4) for rat liver. After removal of the nuclei and unbroken cells, the supernatant was filtered through glass wool and centrifuged for 5 minutes a t 5900g. The pellet consisted of three well-defined layers. The upper pinkish and the middle buff-colored layers were removed, and the lower dark brown layer was suspended in 0 . 6 M sucrose and centrifuged for 5 minutes at 59009. Any light-colored material on the surface of the pellet was removed, and the lower pellet was suspended in 0.6 M sucrose. This suspen-

ACID PHOSPHATASE

87

sion was contaminated to some extent with mitochondrial and microsomal fractions, as manifested by determination of marker enzyme activities, but electron microscopic examination showed about 95% of intact lysosomes and about 5% of mitochondria. Microsoma1 fragments were seldom visible. Employing the procedure of de Duve et al. (D10) for determining intracellular fractions by centrifugation in 0.25 M sucrose, Wattiauxde Coninck et al. (W2) obtained the following percentage distribution for kidney : nuclear fraction, 20.9; heavy mitochondrial, 33.9; light mitochondrial, 7.6; microsomal, 19.9; final supernatant, 15.3. The sum of the acid phosphatase activities in the light mitochondrial and microsomal fractions, in which it is presumed that most of the lysosomes should be gathered, was 27.5%) as compared with 60.8% obtained by the same method for liver (D10). The absolute total acid phosphatase activity for all fractions of the kidney was 5.53 units/g and th a t for liver was 6.06 units/g (D10).

4.3.5. Prostate (Bull,R a t ) In man and in other mammalian species, the major mass of the prostate, usually consisting of the right and left lateral and the middle lobes, is composed of alveoli lined with columnar epithelium embedded in a thick fibromuscular stroma. These alveoli constantly secrete a fluid which is drained off by a system of branching ducts that empty into the floor and lateral surfaces of the posterior urethra. The normal secretion is dependent upon the degree of androgenic stimulation and amounts to about 0.5-2 ml per day. The prostatic secretion, which is characterized by very high acid phosphatase activity, is a milky fluid which contains citric acid, choline, cephalin, cholesterol, proteins, and electrolytes similar to those found in the plasma. Attempts to determine the intracellular distribution of acid phosphatase in the prostate must take into account the presence of this enzyme in the extracellular secretion. Employing centrifugal methods, Siebert et al. (S20) found that of the total acid phosphatase present in bull prostate homogenate, 0.7% was in the nuclear fraction, 41% in the mitochondrial fraction which presumably included the lysosomal component, and 84% in the microsomal and supernatant components. The finding that the sum of these activities exceeded that in the homogenate was considered to represent removal of inhibitors during separation of the fractions. The intracellular distribution of acid phosphatase in the ventral prostate of the rat has also been investigated by Bertini and Brandes (B15). Groups of male rats of two weight levels, 350 k 20g and 180 4 10 g

88

OSCAR BODANSKY

were fasted overnight and sacrificed by exsanguination. The ventral prostates were homogenized in three times their weight of ice-cold 0.35M sucrose, 0.001 M EDTA. The pellet resulting from centrifugation at 6008 for 10 minutes represented the nuclear fraction, N. The supernatant, cytoplasmic fraction, C, was centrifuged at 10,OOOg for 3 minutes to yield the mitochondrial fraction, M ; a light mitochondrial fraction, L, was obtained by centrifugation at 41,0009 for 6 minutes and 40 seconds; a microsomal fraction, P, was obtained by centrifugation a t 105,OOOg for 30 minutes, The resulting supernatant was designated S. Total enzyme activity was elicited by freezing and thawing 7 times in an acetone-dry ice mixture. Sodium P-glycerophosphate was used as the substrate for determination of total and free acid phosphatase activity. In 350-g rats, the distribution of this enl;yme activity, expressed as percent of the sum of the activities in the nuclear and cytoplasmic fractions, was: nuclear 11.3; mitochondrial, 21.8; light mitochondrial, 19.8; microsomal, 10.5; supernatant, 35.1, with a recovery of 99.8%. No substantial difference was found for the 180-g rats, except for a slight increase of latent acid phosphatase. The free acid phosphatase actiyities expressed as percentage of the total acid phosphatase activity for each of the fractions were: nuclear, 53; mitochondrial, 33; light mitochondrial, 55; microsomal, 127; supernatant, 101. The sum of the free activities of all fractions was 71% of the total activity. The comparable values for the fractionation of rat liver (D10) were: nuclear, 60; mitochondrial, 18; light mitochondrial, 12; microsomal, 50; supernatant, 106. The sum of the free activities of all fractions was 22% of the total activity. Thus the latent activity of acid phosphatase in the prostate was less than in the liver. Pellets separated from nuclei-free homogenates in 0.25 M sucrose by centrifugation a t 45,OOOg for 6 minutes and washed twice by resuspending in excess 0.25 M sucrose were analyzed by isopycnic gradient centrifugation. The sucrose concentrations ranged in density from 1.20 to 1.145. The activity curves for acid phosphatase and cytochrome oxidase and the concentration curve for protein showed essentially the same distribution, with a peak a t a density of about 1.180. These results as well as those on the latency of acid phosphatase indicate the possibility that the lysosomes containing the enzyme become disrupted during fractionation or during isopycnic gradient centrifugation, and that acid phosphatase may be absorbed to other subcellular particles in a nonspecific manner. Electron microscopic studies of rat prostatic epithelium, showed acid phosphatase to be present largely in localized areas of the cytoplasmic matrix, lacking in many cases 'structures which would be comparable

ACID PHOSPHATASE

89

with that of the “sacs” having a single-layered, lipid membrane, such as had been described in liver parenchymal cells (B29, B30, H1, H4). Koenig (K10) has raised the question whether the lysosomal enzymes may not, a t least in some tissues, be conjugated ionically with acidic glycolipids in a solid complex.

4.3.6. Testis and Semen The intracellular distribution of acid phosphatase in the testis has only recently been studied (R9). The following fractions were obtained from a homogenate of the testes of adult Swiss mice in 0.25 M sucrose: nuclear, 6009 for 5 minutes; heavy mitochondrial, 10,3009 for 3 minutes; light mitochondrial, 41,OOOg for 7 minutes; microsomal, 105,OOOg for 30 minutes ; supernatant of the last centrifugal procedure. This supernatant fraction contained 65% of the free, uninactivated acid phosphatase, whereas the light mitochondrial fraction, usually presumed to include the lysosomes, contained only 19% of the free acid phosphatase. Activation was accomplished by freezing and thawing of the testicular tissue five times. The activity of the homogenate increased from 6.6 to 9.2 pg P liberated per minute per milligram of protein. As was to be expected, no activation was apparent in the supernatant fraction, but th a t of the light mitochondrial fraction increased from an activity of 1.27 to one of 3.49 pg P liberated per minute per milligram protein, and constituted 38% of the postactivation total acid phosphatase activity. The character of the intracellular distribution of acid phosphatase in the testis appears to differ from that in the kidney or liver, where a major portion of the enzyme is in the lysosomal fraction. Although the finding in the testis may represent a difference in cellular organization, the possibility also exists that the acid phosphatase may be less firmly bound to the lysosomal structure in the testis than in the liver and may be more readily solubilized during the process of homogenization. As long ago as 1935, Kutscher and Wolbergs (K12) observed th a t semen and the prostate are among the richest sources of acid phosphatase in the human body. I n a more recent survey (B11) the acid phosphatase activities of seminal plasma in various species, determined as milligrams of nitrophenol liberated by 100 ml seminal plasma from 0.006M p-nitrophenyl phosphate, in 60 minutes a t 37°C and pH 4.9 were: human, 274,000; cock, 15,000; turkey, 4000; bull, 570; rabbit 85. Human seminal plasma is made up by the secretory fluids produced in the epididymides, vasa deferentia, ampullae, seminal vesicles, the prostate and the bulbourethral (Cowper’s) and urethral (Littre’s) glands (M4). The semen contains many particulate bodies. Best known, of course, are the spermatozoa, which are formed in the seminiferous

90

OSCAR BODANSKY

tubules of the testis and remain in the epididymis for a “ripening” period before ejaculation. Dingle and Dott (D12) have noted that certain lysosomal enzymes, including acid phosphatase, in bull and ram semen are sequestered into membrane-limited droplets, which are shed from the spermatozoon during maturation but persist in the seminal plasma. I n bull semen, these droplets were separated and found to contain acid phosphatase, p-glucuronidase, acid protease and ribonuclease whereas other lysosomal enzymes, such as P-N-acetyl glucosaminodase, p-galactosidase, and hyaluronidase were present predominantly in the spermatozoa. Dott and Dingle (D14) have determined the total, free and bound acid phosphatase of whole semen. Bull semen was diluted 1:20 with buffered saline, and a sample was treated with detergent and the total acid phosphatase activity determined with p-nitrophenyl phosphate as substrate. The activity was 3510 units (pg nitrophenol liberated per hour per milliliter of semen). The remainder of the semen was sedimented a t 10009 for 5 minutes and then a t 10,OOOg for 10 minutes. The combined activity of the peIlets, after washing, was 750 units or 21% of the total and represented the bound activity, that is, the activity in the cells and droplets. The enzyme activity in the 10,OOOg supernatant represented the free or nonsedimentable enzyme (2760 units). In the ram, the values were quite different: 6500 units for the total, 1600 units for the free or nonsedimentable acid phosphatase and 4900 units or 7576 of the total activity for the bound enzyme in the cells and droplets. The cytoplasmic droplet in bull and ram serum is spherical with a diameter of approximately 3 p. When viewed by phase contrast microscopy, it has a dark “granulated” region; electron microscopy reveals that this region consists of vesicles and membranous or tubular structures. The concentration of acid phosphatase in droplets is 4.3 pg nitrophenol liberated per hour per lo6 particles, much higher than the activity, 0.2 pg nitrophenol liberated per hour per lo8 spermatozoa. Ribonuclease, acid protease, P-glucuronidase showed similar ratios. Dott and Dingle (D14) submitted additional information which indicated that in the bull and, to a lesser extent in the ram, the lysosomal enzymes cease to be associated with the spermatozoon during its maturation. 4.3.7. Leukocytes and Macrophages Peritoneal exudates, containing large numbers of polymorphonuclear leukocytes, may be produced in rabbits by the intraperitoneal injection of 200 ml of 0.1% glycogen (C7). Leukocytes from the exudate were withdrawn 4 hours later, washed once in cold 0.34.M sucrose, and

ACID PHOSPHATASE

91

then lysed in the same medium. The sucrose lysate was separated into three fractions by differential centrifugation: the nuclear pellet a t 400g for 10 minutes; the granule pellet at 82009 for 15 minutes; the resulting supernatant. Acid phosphataee as well as several other enzymes, alkaline phosphatase, nucleotidase, ribonuclease, deoxyribonuclease, and p-glucuronidase, were predominantly localized in the granule fraction. The characteristics of acid phosphatase in leukocytes will be discussed further in Section 6.9. I n a subsequent study, Cohn and Wiener (C8) considered the particulate hydrolases of macrophages. Peritoneal macrophages were induced in rabbits by intraperitoneal injection of oil, and aveolar macrophages were obtained from the lungs of normal rabbits or of rabbits following intravenous injection of BCG. The cells were centrifuged, washed, and assayed. The acid phosphatase activity, expressed as micrograms of P liberated from sodium /I-glycerophosphate per hour a t 38" by lo6 macrophages was: 2.6 for oil-induced peritoneal macrophages, 20.7 for normal alveolar macrophages, and 37.0 for BCG-induced alveolar macrophages. The corresponding activities per milligram of N were 118, 600, and 1073. Differential centrifugation of homogenized oilinduced macrophages showed that the acid phosphatase, as well as other hydrolases, was localized as follows: about 20% in the nuclear fraction (500g for 12 minutes), approximately 65% in the fraction sedimented by centrifugation a t 12,0009 for 15 minutes, and the remainder, about 1&15%, in the supernatant fraction. Most of the acid phosphatase activity as well as that of the other hydrolases was latent, as repeated freezing and thawing or a longer time period for assay increased the activities greatly. 4.4. DIGESTIVE FUNCTION OF LYSOSOMES

The importance of lysosomes in physiopathological autolysis, intracellular digestion and engulfing processes was pointed out by de Duve (D7, D8) and Novikoff (N5). Within recent years several studies have appeared in which the actions of purified lysosomal preparations on proteins, carbohydrates and lipids have been considered (A14, C5, M3, S2). Sawant et at. (52) studied the digestion of rat liver homogenate, mitochondria, microsomes, and nuclei by a purified preparation of lysosomes. A few values, particularly for inorganic phosphate, may be quoted to show the extent of digestion a t 37°C and p H 7.0. With regard to the action on liver homogenate, 1 mg of lysosomal protein formed 28 and 38 nmoles of amino acids and peptides in 0.5 hour and 3.0 hours,

92

OSCAR BODANSKY

respectively. Under the same conditions 380 and 840 nmoles of inorganic phosphate were formed in these periods. Similar results were obtained by the action of lysosomes on isolated mitochondria and microsomes. Negligible effects were obtained for the action on nuclei a t p H 5.0. Degradation of mitochondria was influenced by the concentration of lysosomes, pH, and temperature of the reaction system. The formation of inorganic phosphate was, of course, a manifestation of phosphatase action. In this connection, two features were of interest. First, in the mitochondria1 degradation by lysosomes, the rate of formation of inorganic phosphate decreased from values of about 2 pmoles in 30 minutes a t pH of 4-5 to a value of about 0.6 pmole in 30 minutes a t pH 6.0, then rose again to about 2 units a t pH’s of 7 to 9. Second, tartrate and fluoride were potent inhibitors; 2.5 X M tartrate decreased the rate of release to 21% of the control value, and 1X M inhibited the formation completely. Fluoride had a similar effect. 5.

Polymorphism of Acid Phosphatase in Human Erythrocytes

5.1. INTRODUCTION We have already discussed the properties of human erythrocytic acid phosphatase (Section 3.3), and we pointed out that, like acid phosphate in other tissues, it may exist in several isoenzymatic forms. I n 1963, Hopkinson et al. (H13) subjected hemolysates of human red cells from an English population to horizontal starch-gel electrophoresis for 17 hours a t 5°C. The gels were then sliced horizontally, covered with 0.05 M phenolphthalein sodium diphosphate at pH 6.0, and allowed to incubate for 3 hours a t 37°C. Five different electrophoretic patterns of acid phosphatase activity could be distinguished in different individuals. Shortly thereafter Lai and his associates (L2) confirmed these findings and discovered an additional sixth pattern which had been predicted by Hopkinson et al. (H13). The distribution of these patterns in various types of population was assiduously pursued within the next several years, and several new ones were discovered in Negro populations (G3, K2). It is of interest that within several years after the observations of Hopkinson et al. (H13), other human erythrocytic enzymes such as phosphoglucomutase, glucose 6-phosphate dehydrogenase, phosphogluconate dehydrogenase, adenylate kinase, peptidase, and adenosine deaminase were explored intensively with respect to their polymorphism (H2, H11). However, we shall concern ourselves here only with acid phosphatase.

93

ACID PHOSPHATASE

5.2. ELECTROPHORESIS The starch gel electrophoretic patterns obtained by Hopkinson (H11) and his associates (H14) and subsequently by others in Harris’ group (H2) and elsewhere in the world (G3, K2) are shown in Fig. 1. Initial studies by Hopkinson et al. (H13) on 139 randomly selected English males and females revealed the existence of five patterns with the following frequencies in the population: type A, 10.1% ; type BA, 46% ; type B, 34.5%; type CA, 36%; type CB, 5.8%. On the basis of genetic considerations, they predicted the existence of a sixth type, C. Lai (Ll) and his associates (L2) obtained a different distribution in a Brazilian population, but reported evidence for the existence of the type C electrophoretic pattern. Additional studies by Hopkinson (H11) modified the incidences of the patterns slightly: A, 13%; BA, 43%; B, 36%; CA, 3%; CB, 5 % ; C, 0.016%. In their original studies, Hopkinson et al. (H13) had employed a 0.0025 M succinic acid-0.0046 M Tris buffer, pH 6.0 for their gel preparations and a 0.041 M citric acid/NaOH buffer, pH 6.0, as a bridge solution. Using vertical electrophoresis, a mixture of 10.2 ml of formic acid (90%) and 9.25g of NaOH per liter as the bridge buffer, pH 5.0, and a 1 : l O dilution of this to make up the gels, Giblett and Scott (G3) discovered a new electrophoretic pattern, designated as RA, in the red cell hemolysate of a Seattle Negro woman. This and two related patterns, RB and RC, were characterized by a pair of relatively fast moving components together with either typical A or B or C zones.

--

Origin-

Phenotype

A

Postulated pap” genotype

BA papb

B pbpb

--

0.0 CA paPC

CB pbp

C p’pc

0

RA RB BD pop‘ pbpf pbpd

Fm. 1. Diagram of electrophoretic patterns of the several red cell acid phosphatase phenotypes. After Hopkinson (R11).

94

OSCAR BODANSKY

Still another electrophoretic pattern, BD, was observed in a Texan Negro (K2). 5.3. GENETICS These different electrophoretic patterns or phenotypes reflected the possible existence of alleles, or contrasting genes situated a t the same locus in homologous chromosomes. Study of the acid phosphatase patterns or phenotypes in 440 families and in their 925 offspring indicated that the various phenotypes were determined by three alleles, Pa, Pb, and P" and that phenotypes A, B, and C have the homozygous genotypes Papa,PbPb,and PP', respectively. Similarly, phenotypes BA, CA, and CB corresponded to the heterozygotes Papb,PaPC,and PbPc,respectively (H11). The distributions of phenotypes observed in the children were not significantly different from the expected Mendelian proportions. For example, in the data presented by Hopkinson (H11) and his associates, there were 94 matings of parents, each of whom had patterns of BA and therefore had the heterozygotic genotype, Papb.Of the 185 children, one-fourth, or 46, should have had the phenotype A, one-half, or 92, should have had the phenotype BA, and one-fourth, or 46, the phenotype B. The incidences actually found were 40, 91, and 54, respectively. Gene or allele frequencies may be derived from the distribution of various phenotypes among a number of individuals. For example, in a study of 1010 individuals in England, the gene frequencies for acid phosphatase were: Pa, 0.36; Pb, 0.59; P", 0.05. Population data from other countries have accumulated rapidly since Hopkinson's original studies, and some of these may be noted briefly (Table 4). It may be seen that the gene frequencies for the United States for persons of European origin were essentially the same as those in England (G3). For Australians ( L l ) and South Africans (H11) of European origin, the gene frequencies of Pa were 0.33 and 0.32, respectively, somewhat lower than those for England and the United States; the gene frequencies of Pb were somewhat higher, 0.64 and 0.62, respectively. I n Negro populations, regardless of their geographic location, the incidence of Pa was strikingly lower and that of Pb was correspondingly higher (B31, G3, H11, K2). The most extreme deviation from average gene frequencies was observed in a group of 140 Tristan Da Cunhan islanders with a Pa value of 0.09 and a Pb value of 0.91. The Alaskan Eskimos and Atabascan Indians show unusually high values for the frequency of the Pagene, 0.56 and 0.67, respectively ( S l l ) . Recent studies have amplified the essential features of the genetic studies that we have just considered. Wyslouchowa (W12) found the

95

ACID PHOSPHATASE

TABLE 4 GENEFREQUENCIES UNDERLYINQ RED CELLACIDPHOSPHATASE POLYMORPHISM IN VARIOUS POPULATIONS" ~

Geographical location England U.S.A.: European origin U.S.A.: Negro, Ann Arbor U.S.A.: Negro, Seattle Australia: European origin New Guinea: Trobriand South Africa: European origin South Africa: Cape Colored Nigeria: Yoruba a

~~

Number of individuals tested

1010 193

224 363 260 484 99

174 129

Gene frequencies Pa

Pb

Po

0.36 0.39 0.17

0.59 0.55 0.82 0.76 0.64 0.79 0.62 0.70 0.83

0.05 0.06 0.01 0.015 0.03 0.01 0.06 0.02

0.23

0.33

0.20

0.32 0.28

0.17

-

Pr

-

-

-

0.010

-

-

Based on review of data by Hopkinson (H11).

following gene frequencies in a group of 1064 Poles: Pa. 0.319; Pb, 0.585; Po,0.096. These are not essentially different from those reported for English and other European populations (H11). I n his study of Danish populations Lamm (L3) discovered a family with the rare Pd allele which had hitherto been observed only in Negro families (G3, K2). Herbich et al. (H6) studied the pedigree of a family in which unusual segregation of the acid phosphatase phenotypes occurred. In generation 11, the father (11. 7 ) , a heterozygous type CA, and the mother (11. 8 > , a homozygous type B, had a child (111. 1) wit.h a normal type C pattern, indicating a Po allele. In other words, the mother did not appear to have transmitted a Pb allele to her son. Again when the parents (I. 8 and I. 9) of the mother (11. 8) were tested, her father had a type A phenotype and the mother a type BA phenotype. Thus here, too, the father did not appear to have transmitted the expected allele, Pa, to his offspring, and the type B pattern exhibited by 11. 8 was presumably determined by a Pb allele transmitted to her from the heterozygous mother, I. 9. These and other findings in this pedigree suggested that there existed in this family a rare "silent" acid phosphatase allele, Po, such that PaPo individuals were phenotypically A, PbPo individuals phenotypically B, and P"Po individuals phenotypically C. An interesting role of acid phosphatase alleles on an unusual condition in man has recently been reported by Bottini et al. (B27). It has long been appreciated that subjects with erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency may have a severe hemolytic crisis after ingestion of fava beans. Although this deficiency is a necessary condition for the occurrence of hemolytic episodes, not all, indeed only

96

OSCAR BODANSKY

about 30% of subjects with G-6-PD deficiency, exhibit clinical favism. Bottini et al. (B27) observed that the frequencies of Pa and P" alleles of the gene for erythrocytic acid phosphatase in a group of Roman males with favism were 0.355 and 0.109, respectively, and were significantly higher than the corresponding frequencies, 0.261 and 0.080, in a group of normal Roman males. A similar relationship held for Sardinian males with favism, but not for females with this condition. This observation indicates that alleles of a gene coding for an enzyme polymorphic in all human populations affect the fitness of the involved phenotypes in special genotype (G-6-PD deficiency) and nongenotypic conditions (ingestion of fava beans). 5.4. QUANTITATIVE DISTRIEUTION The phenotypes of erythrocyte acid phosphatase not only exhibit differences in electrophoretic behavior, but also show variation in total acid phosphatase activity. Spencer et al. (526) studied the distribution of red cell phosphatase activities in hemolysates from 275 individuals with various phenotypes. The assay was performed with 0.01 M disodium p-nitrophenyl phosphate as substrate at pH 6.0 in citrate buffer. The units of activity were expressed as pmoles of p-nitrophenol liberated in 0.5 hour at 37°C per gram of hemoglobin. These results are shown in Table 5. If it is assumed that all the acid phosphatase activity observed in the various types is determined by the three genes, Pa, Pb, and P" then the question arises whether the quantitative effects of these genes are additive in a simple way. It may be seen from Table 5 that half the mean activity, 122.4, in type A plus half the mean activity, 188.3,in type B is equal to 61.2 94.2 or 155.4. This value is in good agreement with 153.9, the mean value actually observed for type BA. Other equations

+

TABLE 5 ACID PHOSPHATASE ACTIVITYIN VARIOUS Phenotype ~~~~

Number of individuals

PHENOTYPES"

Mean activityb (units)

Standard deviation (units)

122.4 153.9 188.3 183.8 212.3

16.8 17.3 19.5 19.8 23.1

~

A BA B CA CB

33 124 81 11 26

Based on data of Spencer et al. (526). Expressed as micromoles of p-nifxophenol liberated in 0.5 hour at 37°C per gram of hemoglobin. a

b

97

ACID PHOSPHATASE

based on the additive hypothesis give calculated values in accord with those actually observed. It is thus possible to assign activity values to the various alleles: 61 for Pa;94 for Pb; 120 for Pc. Hopkinson (Ell) subsequently enlarged the series to 336 individuals, but the results were essentially the same as those that have just been described. 5.5. BIOCHEMICAL CHARACTERISTICS OF PHENOTYPES

Differences in the electrophoretic patterns and activities between the phenotypes raised the possibility that they might also be characterized in a more specific and detailed biochemical manner. I n 1949, Abul-Fad1 and King (A4) observed that 0.5% formaldehyde inhibited erythrocyte acid phosphatase completely, whereas 0.01 M L- ( )-tartrate had no effect. When 0.5% formaldehyde was added to the reaction mixture in gel electrophoresis (H13), it completely inhibited all variants of erythrocyte acid phosphatase, so that no zones of activity in any of the five types were visible after the 3-hour incubation. Scott (S10) and later Luffman and Harris (L13) failed t o find any kinetic differences between the variants. Scott (SlO) purified the acid phosphatases from two homozygous, phenotypically different, human red cells. These were designated as AA and BB and corresponded to A and B, respectively, in the terminology of Hopkinson et a2. (H13). Employing several different substrates and sodium acetate as buffer, with a final reaction p H of 6.5, Scott (S10) failed to find any differences between the Michaelis constants of these two isozymes; with phenyl phosphate as buffer, the K,,, values were 0.87 and 0.75 mM for the AA and BB phenotypes, respectively. With a-glycerophosphate as substrate, the corresponding K,,, values were 5.8 and 5.5 mM. Nor were any differences observed when p-nitrophenyl or phenolphthalein diphosphate were the substrates. A few small differences could be elicited. Thus, phosphate inhibited the AA variant more than the BB variant. The maximum velocity of the AA variant was relatively lower at lower pH. However, these findings did not account for the finding that the AA variant had 65% of the activity of the BB variant. Luffman and Harris (L13) applied several other criteria in a n attempt to differentiate among the various phenotypes. Incubation of hemolysates representing these phenotypes showed that a t temperatures of 47°C to 52°C types CA and CB were denatured more slowly than the other types tested, A, BA, and B. For example, after 30 minutes a t 50"C, the average losses in activity were: 52% for CA and 57% for CB, as compared with losses of 89% for A, 83% for BA, and 80% for B. Incubation with guanidine or urea a t 28°C for 20 minutes showed denaturation to be dependent on the concentration, but there were no

+

98

OSCAR BODANSKY

differences in the rate of denaturation between these phenotypes. Each of the variants exhibited substantial phosphotransferase activity. For example, in the presence of 20% methanol, the phosphotransferase activity was approximately 300% of the hydrolytic activity in the absence of methanol, but again the phosphotransferase activity was essentially the same for all the phenotypes tested. The rates of hydrolysis of 14 different phosphate esters as substrates were determined, and although the patterns relative to the rate of hydrolysis of p-nitrophenyl phosphate were of interest, there appeared to be no significant differences between the patterns for the various acid phosphatase types. Samples of the four variants, A, BA, B, and CB were subjected to gel filtration together with several substances of known molecular weights as markers. I n every instance, all the acid phosphatase activity emerged from the column as a single peak subsequent to the elution of myoglobin (molecular weight of about 17,000)and cytochrome c (molecular weight of about 12,400).This finding suggested that erythrocytic acid phosphatase may have a very low molecular weight in the region of 700010,000.There were no differences in the elution positions of the enzyme in the different phenotypes (L13). The column chromatography of the five common phenotypes were also studied by Hopkinson and Harris (H12).A 10-ml aliquot of the supernatant from a centrifuged hemolysate was applied to the DEAE column which had been washed with Tris-phosphate buffer (pH 8.0). The column was then washed with the starting buffer to elute the hemoglobin. The enzyme was eluted with an exponential gradient of sodium chloride in Tris-phosphate buffer and collected in 2-ml fractions a t a flow rate of 20 ml/hour. Two distinct peaks of acid phosphatase activity were detected in each phenotype, but the positions of these peaks differed. For example, in phenotype A, the peaks were approximately a t tubes 150 and 190; in B, a t about 130, 170, and 265; in BA, a t 110 and 155. I n these three, the first peaks showed minor enzyme activity. I n CA, there was a major peak a t about tube 130 and a smaller one a t about tube 170. The shape of the curves varied according to the phenotype tested. I n general, these results confirmed what gel electrophoresis originally showed, namely, that there are charge differences between the various isoenzymes. The electrophoretic patterns may also be influenced by the type of buffer used to make up the starch gel (K2). IN OTHER TISSUES 5.6. POLYMORPHISM The question arises whether polymorphism can be demonstrated for the acid phosphatases of other human tissues. Beckman and Beckman

ACID PHOSPHATASE

99

(B10) carried out starch gel electrophoretic studies in 1200 individual placentas and in extracts of seven different organs obtained a t autopsy from 14 individuals. The tissues showed different combinations of one or more bands of four distinct and, in some respects, biochemically different, acid phosphatase components. These were designated as A, B, C, and D in order of decreasing anodal mobilities. For example, in the case of heart tissue one individual showed three bands, ABD, whereas the remaining 13 showed a combination of two bands, BD. With regard to kidney tissue, 13 individuals had a combination of ABD, and one individual had a pair, BD. The C component was present in all extracts of the 1200 placentas, but in none of the other organs. The typical placental combination was BCD. Three out of 1000 placentas showed a deviating electrophoretic pattern. The B zone was not affected. In addition to the usual C component, there was another somewhat slower component. The D zone contained three components of which the fastest one coincided with the normal D band. This deviating pattern in the three placentas probably represented genetically determined variants. 6.

Alterations of Serum Acid Phosphatase Activity in Disease

6.1. INTRODUCTION At the beginning of this review, we noted that acid phosphatase activity was first found to be present in human urine (D11). Approximately ten years later, Kutscher and his associates (K11, K12, K13) described its presence and properties in the various organs of the male genital tract and in other tissues. The clinical significance of this enzyme in prostatic disease was elicited by Gutman and his associates (G11, G12, G13, R6, 530). Subsequent investigators not only extended these findings, but also sought to correlate alterations in serum acid phosphatase activity with treatment of various types, to determine whether alterations in nonprostatic disease might also occur and to examine basic mechanisms involved in these alterations. Since different methods were employed in these various studies, we shall describe the results of these studies in some detail. ACTIVITY 6.2. NORMAL VALUESFOR SERUMACIDPHOSPHATASE The method of Gutman and Gutman (G10, G14), an application of the King-Armstrong (K5) method for alkaline phosphatase to acid phosphatase, was described earlier in this paper. The activity by this method was defined in units, as the number of milligrams of phenol liberated in 1 hour a t 37°C by 100 ml of serum. These have been fre-

100

OSCAR BODANSBY

TABLE 6 VALUESFOR SERUMACIDPHOSPHATASE XN NORMAL ADULTMALESBY METHOD OF GUTMANAND GUTMAN (G10, G14) OR SLIQHT MODIFICATIONS OF IT

THE

K.A. unitsa Author Gutman and Gutman (G10) Sullivan et al. (S30) Fishman and Lerner (Fl) Fishman et al. (F2) Day et al. (D6) Day et al. (D6) Benotti et al. (B12)

Number of individuals 10 30 13 104 136 (20-39 years old) 179 (40-79 years old) 22

Range

Mean

Standard deviation

0.6-2.0 <3.0 0.7-1.7 <5.0 1.14.7

1.2

0.39

1.2 1.8 2.70

0.34 0.8 0.57

0.84.0

2.47

0.56

1.2

1.2

-

-

-

* Units are equal to milligrams of phenol liberated from phenyl phosphate in 1 hour at 37°C by 100 ml of serum.

quently referred to as King-Armstrong (K2A.) units. Table 6 shows various series of values obtained with this method in normal males. Values on large groups of patients with nonprostatic disease have not been included. Some of these results were obtained in connection with the proposal of a procedure for the determination of the “prostatic” component of acid phosphatase in serum by the use of the inhibitor, L-(+)-tartrate (Fl). We shall discuss this aspect in greater detail later. The determination of the range of acid phosphatase activity in apparently healthy normal males must naturally take into account the effect that benign prostatic hypertrophy may have. This problem was considered by the writer and his associates in 1956 (D6). Utilizing the Gutman procedure (G10, G14), together with the modifications Fishman and Lerner ( F l ) had proposed, the “total” and “prostatic” moieties of acid phosphatase were determined in a large group of male individuals who had presented themselves a t a cancer-prevention clinic for general examination. Of a group of 141 men from 20 to 39 years of age, five had some degree of prostatic enlargement. The remaining 136 had serum acid phosphatase activities ranging from 1.1 to 4.7 units with a mean value of 2.70 K.A. units and a standard deviation of 0.57 unit (Table 6). In this group, there were four men with acid phosphatase values higher than 3.8. In the five individuals who had clinically abnormal prostates but no evidence of prostatic carcinoma, the serum acid phosphatase activities were all normal, this is, less than 3.84 units (2.70

ACID PHOSPHATASE

101

plus two standard deviations). I n a group of 119 males, 40-79 years of age, with clinically normal prostates, the serum acid phosphatase activities ranged from 0.8 to 4.0 K.A. units and averaged 2.47 K.A. 2 0.56 K.A. units. There were five individuals with acid phosphatase values greater than 3.8 K.A. units. In this same age group, there were 46 males who had enlarged prostates but only four of these had values greater than 3.84 units: 4.1, 4.2, 4.2, and 4.7 units. Thus i t would appear that in this total group of 366 males, neither age nor benign prostatic enlargement had an effect on acid phosphatase activity; 96% of the values were less than 2 standard deviations above the mean, and the remaining individuals had acid phosphatase values between 2 and 3 standard deviations above the mean value. The mean normal values listed in Table 6 are not, in general, different statistically from each other. The value of 2.7 k 0.57 (D6) appears significantly higher than that calculated for the group of 13 subjects reported by Fishman and Lerner ( F l ) , 1.2 k 0.34 K.A. units, even though Fishman and his co-workers stated elsewhere (F2) that normal values ranged from 0.5 to 5.0 units. It is difficult to state whether these differences among different series are due to variations in the nature of the population or in methodological differences, such as spontaneous hydrolysis in the substrate (B12) or the loss in acid phosphatase activity that a serum undergoes when it is separated from the clot and allowed to remain at room temperature for varying periods of time before analysis (W7). The next most common method for determination of serum acid phosphatase activity was based on the use of sodium p-glycerophosphate as substrate. This method and its modifications have been described in Section 2.3. Values obtained by these methods in normal males are shown in Table 7; a liberal summary of these values indicates a mean value of about 0.4 -+. 0.2 Bodansky units. 6.3. SERUM ACIDPHOSPHATASE IN CARCINOMA OF THE PROSTATE

Carcinoma of the prostate is today one of the three most frequent causes of death from neoplastic disease in men in the United States (G4). The early studies of Gutman and his associates (G11, G12, R6, 530) established that serum acid phosphatase activity was elevated very frequently in patients with metastatic carcinoma of the prostate. It is of interest to consider briefly the uncertainties inherent in the development of this relationship. Skeletal metastases, if sufficiently large, are of course detectable by roentgenographic examination, but smaller ones may not be, and metastases to soft tissues may likewise be undetectable. For example, in 15 cases of metastazing carcinoma reported by Gutman

102

OSCAR BODANSKY

TABLE 7

VALUESBOR SERUMACIDPHOSPHATASE IN NOWL MALESBY THE METHOD OF A. BODANSKY (52) OR SLIQHT MODIFICATIONS THEREOF

Investigator

Bodansky (B19) Shinowara et al. (S18) Woodard (W8, W9) Marshall and Amador

Upper limit of Standard (mean deviation 2 SD)

+

Number ofnormah

Range

Mean

43 20 47 36

0.1-1.1 0.11-0.88 0.1-0.5

-

0.19

0.048

0.45 0.28

0.12

-

-

0.29 1.1 0.88 0.51

035)

and Gutman (G11) in 1938, 12, or 80%, exceeded the normal range of serum acid phosphatase activity, 0.5-2.5 K.A. units, as previously defined by them. The highest was 516 K.A. units, and the lowest was 3.1 K.A. units. But the latter patient, who had extensive osteoplastic metastases, had already been subjected to resection of the prostate. Of the three patients with values of 1.6, 1.5, and 1.5 units, two had questionable or no bony metastases and the third patient had osteolytic and osteoplastic metastases but had been treated by implantation of radium seeds. Shortly thereafter, a second paper from the Gutman group (R6) presented a series of 28 determinations of serum acid phosphatase on 19 patients with roentgenographic evidence of skeletal metastases. These ranged from 1.6 to 260 K.A. units; 23, or 89%, were higher than 3.0 K.A. units, the upper limit of normal, as defined in this study. Thirteen patients with prostatic carcinoma but without any roentgenographic evidence of skeletal metastases had serum acid phosphatase levels of 0.5 to 2.6 K.A. units, all less than the upper limit of 3.0 K.A. units of Sullivan et al. (530). These findings in Gutman’s group are summed up most comprehensively in Table 8. It may be seen that 85% of patients with prostatic carcinoma and skeletal metastases had elevations above their designated upper limit of normal, 3.0 K.A. units. Five patients had sensationally high values, more than 1000 K.A. units. The frequency of elevations in the patients with no bone metastases, as visualized roentgenographically, was 11%.This group was not described in detail, and it is possible that some of the patients had soft tissue metastases or nondetectable bone metastases. The general presumption is that in these cases extracapsular extension of the prostatic carcinoma had not proceeded too far. Sullivan et al. (S30),however, described one

103

ACID PHOSPHATASE

TABLE 8 ELEVATIONS OF SERUM ACID PHOSPHATASE ACTIVITY IN DISEASES OF THE PROSTATE Sullivan et al. (S30)

Disease Carcinoma of prostate with bone metastases Carcinoma of prostate without bone metastases Benign prostatic hypertrophy Prostatitis

No. of patients

Incidence of elevations

(%I

Herbert (H5)

No. of patients

Incidence of elevations

(%I

130

85

35

89

70

11

47

42

75 10

0.0 0.0

95

-

9.5

-

patient who died of congestive heart failure 3 days after a normal serum acid phosphatase determination of 2.4 K.A. units. Autopsy showed a n early, regionally invasive but not distantly invasive carcinoma of the prostate. Table 8 indicates that benign prostatic hypertrophy or prostatitis does not cause any elevations of acid phosphatase. This confirmed early studies by Gutman and Gutman (G11). Reports by others, using the Gutman method (G10, G14), did not quite agree with the preceding results. Thus Herbert (H5), utilizing a normal range of 1 to 4 K.A. units, observed a similar incidence of serum acid phosphatase elevations in prostatic carcinoma with skeletal metastases, 31 out of 35 patients, or 89%, but a much higher incidence of elevations than Gutman’s group in prostatic carcinoma without bone metastases, namely, 20 of 47 patients, or 42%. Four of these patients had substantial elevations, that is, above 10 K.A. units. Herbert (H5) also observed a 9.5% incidence of elevations, slight though these were, in benign hypertrophy of the prostate. There are a number of other studies in the literature in which serum acid phosphatase was determined by the method of A. Bodansky or slight modifications thereof. The important feature of this method is the use of sodium /3-glycerophosphate as substrate. As has been noted earlier, Woodard (W8) established the normal range in 20 normal females as averaging 0.38 unit (milligrams of phosphorus liberated in 1 hour by 100 ml of serum under certain standardized conditions) and ranging from 0.06 to 0.89 unit. The activity in 47 normal males was essentially the same-an average of 0.45 unit and a range of 0.11 to 0.88 unit. Patients with various diseases not involving the bladder, rectum, or prostate had essentially the same average values and range, so that a

104

OSCAR BODANSKY

broad range of “normal” values could be established for 492 patients without prostatic diseases as averaging 0.39 unit, with a range of 0.00 to 0.98 unit. The data of Woodard and Dean (W11) and of Woodard (W8) for patients with prostatic disease are sufficiently comprehensive to represent several other studies based on the use of Na P-glycerophosphate instead of phenyl phosphate as substrate. Thus, in 107 patients with carcinoma of the prostate with metastatic lesions to the bones, the range of serum acid phosphatase activities was 0.10 to 520 Bodansky units, with 74% above the upper limit, 0.98 unit. I n 51 patients where the tumor was locally invasive, the range was 0.0 to 12.9 units, with 31, or 60%, above the upper limit of normal. I n 20 patients with carcinoma confined to the prostate, the range was 0.28 to 5.9 units with 5, or 25@, above the upper limit of normal. In 9 patients with metastases to distant soft parts, the range was 0.72-18.3 units with 78% above the upper limit of normal. Twelve patients with prostatitis and 10 patients with carcinoma of other origins involving the prostate showed serum acid phosphatase values well within the normal range. On the ingenious assumption that early bone metastases might reveal themselves by increased elevations of acid phosphatase in the bone marrow blood, Chua et al. (C3) determined the enzyme activity in 10-ml samples of blood taken simultaneously from a cubital vein and from the area of the posterior-superior iliac spine. The Bodansky method (B17, 52) with P-glycerophosphate as substrate was employed to determine the acid phosphatase activity in the serums from both sites. Four of 12 patients with clinically localized carcinoma of the prostate had elevated bone marrow acid phosphatase with normal serum acid phosphatase levels and skeletal surveys negative for metastases. One of these patients had a strikingly elevated bone marrow acid phosphatase (116 Bodansky units), and a bone marrow biopsy which disclosed the presence of metastases even though there was no radiological evidence of these. Of 13 patients with clinical extension of the cancer beyond the prostatic capsule, seven who were receiving antiandrogens and had undergone bilateral orchiectomy showed normal bone marrow and serum acid phosphatase activities. Of the remaining six patients, one had slightly elevated bone marrow acid phosphatase activity and a normal serum acid phosphatase activity. I n the other five patients, both the marrow and the serum acid phosphatase values were elevated, but the former were higher. Of 13 patients who had osteoblastic changes in the skeletal system, increased acid phosphatase values were obtained in both bone marrow and serum in 10 cases, but the values in the bone marrow

ACID PHOSPHATASE

105

samples were much higher. The results in these three groups indicate that measurement of bone marrow acid phosphatase may offer aid in the early diagnosis of bone metastases from prostatic carcinoma. The importance of defining the normal range before evaluating serum acid phosphatase elevations with prostatic disease is well brought out by the more recent study of Marshall and Amador (M5). Using P-glycerophosphate as substrate and a group of 36 healthy ambulatory males, 41 to 57 years of age, these investigators found a range of 0.1 to 0.5 unit, with a mean of 0.28 If: 0.116 unit and an upper limit (mean + 2 SD) of 0.51 unit. This range was narrower and the mean value lower than those obtained by Woodard (W8, W10) or by Shinowara et al. (SlS), and was much closer to that, 0.19 k 0.048, obtained by A. Bodansky (B18). Employing their own criterion for the normal range, Marshall and Amador (M5) found that 27 of 57 patients, or 46%, with intracapsular prostatic carcinoma and 15 of 27, or 56%, of patients with soft tissue metastases had values above 0.5 unit, the upper limit of normal. However, if Shinowara and his associates’ (S18) value of 1.1 unit had been taken as the upper limit of normal, the incidence of elevations in patients with carcinoma of the prostate and skeletal metastases, soft tissue invasion, or intracapsular confinement would have decreased to 65%, 30% and 16%, respectively. OF SERUMACIDPHOSPHATASE DETERMINATION FOR 6.4. SPECIFICITY CARCINOMA OF THE PROSTATE

6.4.1. Kinetic Considerations Preceding data have already indicated, and subsequent discussion will confirm, the view that the use of P-glycerophosphate as substrate yields a lower incidence of elevations in nonprostatic disease than the use of phenyl phosphate and in this sense constitutes a more specific method. Several authors have distinguished between these two methods by using the terms “acid glycerophosphatase” and “acid phenylphosphatase” activities (B6, T7).This specificity might be explained by the fact that sodium P-glycerophosphate, the substrate in the Bodansky procedure, is hydrolyzed more readily than phenyl phosphate, the substrate in the Gutman procedure, by acid phosphatase from the prostate and that the converse situation holds for enzyme derived from other tissue sources, such as the erythrocyte or the liver. Abul-Fad1 and King (A4) found that in the presence of 0.01 MgZ+,the rate of liydrolysis of 0.02M P-glycerophosphate was 30 mg P per 30 minutes per 100 ml of human prostate extract and was much higher than the rate of hydrolysis, 0.3 mg of P by human erythrocytes. In contrast, phenyl phosphate was

106

OSCAR BODANSRY

hydrolyzed much more readily by erythrocytic acid phosphate, 48 mg of P per 30 minutes per 100 ml, a rate essentially equal to the rate of hydrolysis by prostate. The Michaelis constant, K,, for prostatic phosphatase is much higher with P-glycerophosphate than with phenyl phosphate as substrate. Nigam et al. (N3), employing a purified preparation of human prostate phosphatase, obtained Michaelis constants of 0.75 mM with acetate buffer (pH 5.1) for phenyl phosphate and 4.0 mM for glycerophosphate and corresponding values of 0.09 mM and 2.0 mM with citrate buffer at, pH 4.9. Tsuboi and Hudson (T3) found values for K, of 0.15 mM for phenyl phosphate and 2.4 mM for P-glycerophosphate for a 300fold purified human prostatic preparation, although these investigators noted that the degree of purification had little effect on the value of K,. In addition to the studies cited above, there are several others showing that phenyl phosphate is much more readily hydrolyzed than P-glycerophosphate by acid phosphatase from human erythrocytes, whereas no such marked difference exists with respect to human prostatic phosphatase (B2,T1, T3). Unfortunately, there do not appear to be any systematic investigations of the substratevelocity relationship for the acid phosphatases of other human tissues. In general, the available data would indicate that P-glycerophosphate is a more specific substrate than phenyl phosphate for the detection and assay of acid phosphatase coming from the prostate, 6.4.2. IdiibitiOn by L-(

+ )-Tartrate

Another procedure to increase the specificity of acid phosphatase determinations for prostatic disease has involved the use of L- ( ) -tartrate to distinguish between the enzyme from the prostate and other tissues, In a series of papers from 1947 to 1949, Abul-Fad1 and King (All A2, A3, A4) studied the properties of various acid phosphatases and reported that 0.01 M L- ( + )-tartrate inhibited the hydrolysis of phenyl phosphate by human prostatic acid phosphatase dissolved in normal saline or in plasma to the extent of 95%, but had no effect on the hydrolysis by acid phosphatase from erythrocytes. The inhibitions of acid phosphatases from other human tissues were as follows: liver, 70%; kidney, 80%; spleen, 70%. Several years later, Fishman and his associates (Fl, F2, F3) applied this principle to the determination of the tartrate-inhibitable or prostatic fraction in serum. This method involved the hydrolysis of disodium phenyl phosphate into phenol and phosphate by serum in the absence and presence of 0.02 M L-( +)-tartrate for a period of 1 hour a t 37°C. Suitable blank and control solutions were employed. The activity in the

+

ACID PHOSPHATASE

107

absence of L- ( +)-tartrate represented the “total” acid phosphatase activity. The activity in the presence of 0.02M L-(+)-tartrate was subtracted from the “total” acid phosphatase to denote the inhibited or “prostatic” acid phosphatase. The values for “prostatic” phosphatase in normal subjects ranged from zero to 0.6 K.A. unit (F2). The proposed diagnostic utility of this procedure was based on the assumption that early cases of prostatic carcinoma might have normal values for the total acid phosphatase, but would reveal elevations of the prostatic fraction. In the series of 13 normal males studied by Fishman and Lerner (Fl) to which reference has already been made and in which the serum total acid phosphatase ranged from 0.7 to 1.7 K.A. units (Table 6 ) , the prostatic acid phosphatase component ranged from 0.1 to 0.3 K.A. The following distribution of prostatic acid phosphatase activities was obtained in a series of 151 male patients without prostatic cancer but suffering from other diseases, such as cardiovascular disorders, other forms of cancer, arthritis, and diabetes: 0 K.A. unit, 4% ; 0.1 unit, 37% ; 0.2 unit, 37% ; 0.3 unit, 13%; 0.4 unit, 7% ; 0.5 unit, 2%. The distribution of the corresponding total acid phosphatase activities in this group was: 0.0-0.5 unit, 13.0%; 0.6-1.0 unit, 43% ; 1.1-1.5 units, 35% ; 1.6-2.0 units, 6%; 2.1-3.0 units, 3% ( F l ) . In a group of approximately 100 female patients with the same diseases, the distribution of the prostatic component was understandably shifted to the lower values. Thus 65% of the patients had prostatic acid phosphatase levels of 0.1 unit or less. The corresponding distribution of total acid phosphatase activity was 0-0.5 unit, 7 % ; 0.6-1.0 unit, 33%; 1.1-1.5 units, 39%; 1.6-2.0 units, 14%; 2.1-3.0 units, 9%. Fishman e t al. (F3) reported that in a group of 12 patients with proven prostatic cancer, five patients without evidence of metastases had normal values for total acid phosphatase between 1.3 and 3.0 K.A. units. Yet in four of these the prostatic portion was 0.7 to 2.2 units, all elevated above the normal level of 0.5 unit. These investigators also indicated that early elevations of the prostatic component in the presence of a normal total acid phosphatase might be the herald of progression of disease with ultimate elevations of total acid phosphatase. As was previously noted (Section 6.2), the total and prostatic serum acid phosphatase levels were determined by the method of Fishman and Lerner ( F l ) in a series of 365 males attending a cancer-prevention clinic (D6).The values for the total acid phosphatase activities in the 315 patients of this group who had no prostatic enlargement have already been described (Table 6 ) . I n groups of the size under consideration, values within 2.5 standard deviations of the mean can be considered as normal;

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accordingly, for the group 20-39 years of age, the upper limit of normal for the total acid phosphatase would be 4.13 units and that for the prostatic acid phosphatase would be 0.65 K.A. units, respectively. The corresponding upper limits for the group, 40-79 years of age were essentially the same, namely, 3.87 and 0.66 K.A. units, respectively. Table 9 shows the number of abnormal total and prostatic acid phosphatase values in male individuals who had presented themselves a t a cancer clinic and who on examination showed no benign prostatic hypertrophy (D6). Because of the possibility that, in accordance with Fishman and his associates’ concept (F3), these abnormal values for the prostatic portion might be a forerunner of advancing disease, determinations were repeated after various intervals. The individual with an initial prostatic acid phosphatase value of 1.14 units showed a value of 0.36 units 5 weeks later and a value of 0.30 units 6 weeks after his first visit. Table 9 shows that, in the upper as well as the lower age group of persons with clinically normal prostates, there were essentially no valid elevations in the total or prostatic acid phosphatase. In addition, five individuals in the younger age group (20-39 years) had clinically abTABLE 9 PROSTATIC SERUM ACID PIIOSPHATASE LEVELS IN MALE PATIENTS (WITHOUT PROSTATIC ENLARGEMENT) OF A CANCER PREVENTION CLINICO

Age group

Number

Number of normal total and normal prostatic values

20-39 years

136b

131

40-79 years

1790

171

~~

~

Number of high total and normal prostatic values

2 (4.23/0.06 4.5310.39)

Number of normal total and high prostatic values

Number of high total and high prostatic values

0

3 (3.03/0.69 3.09/0.99 3.84/1.14) 3 5 (3.96/0.39 (3.0910.69 3.99/0.18 3.51/0.75 3.9910.21) 3.5110.81 3.54/1.38 3.81/0.75)

0

~

~~

Based on data of Day et al. (D6). b Normal mean values in this group were 2.70 f 0.57 K.A. units for the total serum acid phosphatase, and 0.14 f 0.205 K.A. unit for the prostatic moiety. Upper limits of normal were 4.13 and 0.65 K.A. units, respectively. 0 Normal mean values in the group were 2.47 f 0.65 K.A. units for the total serum acid phosphatase and 0.15 f 0.204 K.A. unit for the prostatic moiety. Upper limits of normal were 3.97 and 0.66 K.A. units, respectively. Data according to Day et al. (D6). 0

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normal prostates, and four of these had normal values for the total and prostatic acid phosphatases. The fifth had a normal total acid phosphatase value of 3.09 K.A. units, but a value of 1.09 units for the prostatic moiety. In the 224 persons in the older age group, of whom 179 had normal prostates, the incidence of clinically abnormal prostates was much higher, 45, or 26%. Yet 40 of these had normal total and prostatic phosphatases. Of the remaining five, three had high total and normal prostatic acid phosphatases, namely 4.14/0.54, 4.17/0.24 and 4.74/0.18 K.A. units. One had a normal total and high prostatic acid phosphatase value of 3.24/1.02 K.A. units, and the remaining individual had both a high total and high prostatic acid phosphatase value of 4.17/1.98 K.A. units. There were only eleven in the entire series of 365 patients who had high prostatic acid phosphatase values at their first visit. In each of the patients in this group who had subsequent determinations, the values for the prostatic acid phosphatase were normal. For example, the patient in the older group who had elevated values for the total and for the prostatic fraction, namely, 4.17 and 1.98 units, on the first visit had normal values on the second visit 3 months later, namely, 2.85 and 0.39 units, and normal values of 2.85 and 0.06 units 1 week later. Clark and Treichler (a) have studied the psychic factors involved in prostatic secretion, and it is possible that physiological or psychological stimuli might have played a role in yielding high values for prostatic acid phosphatase activity on the first visit. These patients were followed for a period of 6 months to a year and no evidence arose which indicated that these elevations had any pathological significance (D6). In 1956, Fishman et al. (F2) summed up their experience with a series of 91 cases of proven cancer of the prostate and a total of 1198 patients with other diseases. Of these 91 cases, 32, or 3576, had elevated total serum acid phosphatase activities. This incidence was much lower than that, 850J0,reported by Sullivan et al. (S30) in 1942 or the value of 89% reported by Herbert (H5)in 1946 for patients with carcinoma of the prostate and skeletal metastases. These investigators had used the method of Gutman and Gutman (G10, G14), which was essentially the same method as that employed by Fishman and Lerner (Fl) and gave the same ranges of normal values (Table 6 ) . The possibility existed that treated cases had normal total serum phosphatase activities and thus weighted the overall incidence toward a low value. However, the data of Fishman et al. (F2) show that the incidence of total serum acid phosphatase activities in treated cases was 20/52, or 38%, even higher than the incidence 12/39, or 31%, in the untreated caBes. Determinations of the prostatic serum acid phosphatase activity in

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these 91 cases yielded the following incidences of elevated values: 84% in the group as a whole, 87% in the untreated cases, and 81% in the treated cases. Although these values were much higher than those for the incidence of elevations of total acid phosphatase activity in these cases, they were of the same order of magnitude as those reported for the total acid phosphatase activity by Sullivan et aZ. (530) and by Herbert (H5). In the series of 91 cases studied by Fishman et aZ. (F2), the incidence of elevations of total acid phosphatase was 25 of 53 cases or 47% in patients with bone metastases, two of 12, or 16%, in patients with soft tissue metastases and four of 26, or 15%, in patients with no metastases. These values are also much lower than the overall values obtained by Bodansky and Bodansky (B19) in a review of the literature up to 1951, according to which total acid phosphatase was elevated in 81% of 349 cases with bone metastases and in 24% of 218 cases without such metastases. Whatever may be the reason for the low incidence of elevations of total serum acid phosphatase activity in Fishman and his associates’ (F2) series of proven cases of carcinoma, the higher incidence of elevations of prostatic acid phosphatase activity indicates that in the patients of this series the determination of the latter was a more sensitive indicator of the presence of prostatic carcinoma. Moreover, when serum prostatic acid phosphatase activities were determined during the course of a patient’s illness, they paralleled the exacerbation or remission of the disease. Whitmore et al. (W4) have considered the relationship of clinical status to the total and prostatic acid phosphatase activities in 20 patients with proven carcinoma of the prostate. It is of interest to note a report in which a patient who had been operated for a gastric malignancy ten years prior to admission began to show symptoms of metastases, especially to the bone, and upon further study revealed high total and prostatic serum acid phosphatase values (521). The former ranged from 4.7 to 8.1 K.A. units and the latter from 1.8 to 2.3 K.A. units. Various forms of therapy for prostatic carcinoma failed to affect the course of the disease. Postmortem examination yielded neither gross nor microscopic evidence of prostatic carcinoma. 6.4.3. Adventitial Elevations in Serum Acid Phosphatase Activity Massage, palpation, or other trauma or pressure on the prostate may result in sudden elevations of the serum acid phosphatase. Hock and Tessier (H10) observed that prostatic massage caused elevations above the initial value in 17 of 20 patients. The serum acid phosphatase usually

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attained its maximal value within 1 hour after massage and returned to normal levels in 2 4 4 8 hours. The highest value observed in this series was 15.5 Bodansky units, as compared with the normal range of 0.0 to 0.8 unit. Infarcts of a noncarcinomatous prostate gland have been reported to be associated with high serum acid phosphatase (527). Daniel and Van Zyl (D2) observed that ordinary digital rectal palpation of 24 patients with cystic benign hypertrophy was followed by a significant rise of serum acid phosphatase in three of the patients, probably as a result of the rupture of wall cysts and the release of phosphatase-rich secretion into the blood. The author has noted elevations in serum acid phosphatase that appeared to be due to the use of catheters or the formation of fecal impactions. Bonner et al. (B26) also studied in some detail the changes in total and prostatic serum acid phosphatase following prostatic massage. Five patients without a prostate gland showed no alteration in either the total or prostatic acid phosphatase during 2 hours after massage. I n three patients with carcinoma of the prostate, alterations reflected the severity of the disease. One patient had an initial total acid phosphatase value of 40.0 K.A. units and a prostatic phosphatase activity of 37.0 K.A. units. These rose to 46.0 and 42.0 K.A. units, respectively, 0.5 hour after massage. I n a second patient, the total serum acid phosphatase activity rose from 5.4 to 7.4 K.A. units in 0.5 hour, and the prostatic acid phosphatase from 5.2 to 6.4 K.A. units. The third patient in this group had an initial total serum acid phosphatase activity of 1.2 K.A. units and a prostatic fraction of 0.2 K.A. unit. There was no significant rise in either value. 6.5. FACTORS INVOLVED IN ELEVATION OF SERUM ACID PHOSPHATASE IN CARCINOMA OF THE PROSTATE 6.5.1. General Considerations The mechanisms involved in maintaining the level of a serum enzyme in the circulation are largely unknown. Generally, it may be conceived that these levels are the dynamic resultant of several processes: (a) the rate of production of the enzyme by one or more tissues; (b) the rate of secretion of the enzyme by the tissues into the circulation; (c) the extent of damage of enzyme-rich tissue with consequent leakage into the circulation; (d) the degradation of the enzyme in the circulation or its degradation by various tissues as the blood circulates through these tissues; (e) the excretion of the enzyme through the kidney, particularly if the enzyme is of a relatively low molecular weight.

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OSCAR BODANSKY

6.5.2. Tissue Acid Phosphatase Activity in Carcinoma of Prostate and in Metastases from Carcinoma of the Prostate Although the fibromuscular structure of the prostate has some acid phosphatase activity, histochemical studies reveal that most of the activity is present in the epithelial cells of the secreting glands ( R l a ) . In carcinoma of the prostate, the staining of the epithelial cells for acid phosphatase show greater variation. The staining may be less, equal to, or greater than in the normal prostate ( R l a ) . Woodard (W8, W9) showed that the carcinomatous prostate contains less acid phosphatase than the normal gland. Specimens from 19 normal prostates yielded a range of 54-1350 units per gram of tissue and an average value of 478 Bodansky units per gram, whereas analysis of 13 carcinomatous prostates from untreated patients gave a range of 16-600 units per gram, and an average of 258 units per gram. The average values for 12 carcinomas from treated patients was 20 units per gram of tissue and for 26 glands with benign hypertrophy, 775 units per gram. I n contrast to the decrease of acid phosphatase activity generally found in the carcinomatous prostate, metastatic sites show markedly increased activities, as compared with the normal tissue that is the site of the metastases. For example, Gutman et al. (G13) found, in one case, the acid phosphatase activities of osteoblastic metastases were 19.0 K.A. units per gram in the lumbar vertebra and 18.6 units per gram in the rib metastases, as compared with values of 0.1 to 1.3 units per gram at similar normal sites in patients without prostatic carcinoma metastases. Woodard (W9) observed that the primary site as well as metastases in patients with osteogenic sarcoma had low acid phosphatase activities, from 0.09 to 0.85 Bodansky units per gram. In contrast, the activities at metastatic sites in patients with prostatic carcinoma were much higher, frequently about 15 units per gram, and, in one instance, ranged up to 185 units per gram. 6.5.3. Semm Acid Phosphatase during Treatment of Patients with Carcinoma of Prostate I n 1941 Huggins and Hodges (H17) observed that treatment of carcinoma of the prostate by bilateral orchiectomy or by estrogen injection resulted in many instances in clinical improvement of the patient and a concomitant decrease of the serum acid phosphatase activity. They also found that in each of eight patients serum acid phosphatase activity feIl rapidly after bilateral orchiectomy (H17). For example, in one patient, the serum acid phosphatase decreased from 26 K.A. units immediately preoperatively to 5 K.A. units, slightly above the upper limit of normal,

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113

within a period of 7 days after operation. During a subsequent period of observation for 150 days, the serum acid phosphatase fluctuated between 2.5 to 7.5 units. I n a second patient, the serum acid phosphatase decreased from 35 units preoperatively to 3 units within 9 days postoperatively and thereby fluctuated between 3 and 6 units during a subsequent observation period of 104 days. The injection of estrogen had a similar effect. For example, one patient was injected with 30 mg of stilbestrol in 23 days, The serum acid phosphatase decreased from 48 to 4.5 K.A. units and remained a t the latter level for another 10 days of observation. In general, the decreases were not as marked as those reported for patients who had been orchiectomized. The observations by Huggins and his associates (H17, H20) have been generally confirmed since 1941 (F3, H8, 523, W l ) . However, there have been reports, particularly in more recent years, that the level of serum acid phosphatase may not always bear a clear relationship to the apparent clinical progress of the disease or the extent of the metastases at autopsy. This lack of relationship is instructive and may be illustrated by the following two cases studied by Bodansky (B22). The first patient, a 70-year-old man, had had a transurethral resection four years previously and had been diagnosed as having prostatic carcinoma. Orchiectomy was performed, and he was placed on stilbestrol therapy. He remained asymptomatic for approximately two and a half years when pain developed in the hip, back, and both rib cages. No record of previous blood biochemical studies was available, and these were now instituted. During the next 5 months, the serum acid phosphatase remained a t very high levels, fluctuating between about 80 and 130 Bodansky units (normal 0-0.8 unit). Roentgenographic studies revealed widespread osteoblastic and some osteolytic metastases in the spine, ribs, pelvic bones, left femur. Hypophysectomy was performed and the patient was given cortisone therapy and followed closely until his death some 3 months later. The alkaline phosphatase was less than 10 Bodansky units on many repeated occasions and decreased toward a normal level at about a month and a half preceding his death. These relatively low levels indicated absence of any sizable intrahepatic or osteoblastic skeletal metastases. Until the patient’s death, the serum acid phosphatase continued to oscillate between 90 and 130 Bodansky units. It would appear that during the 5 months of study there was no growth or extension of metastases, but rather an active production by these metastases of large amounts of acid phosphatase. A second patient, P.G., 76 years of age, was found in April 1952 to have an enlarged prostate, but only a slightly elevated serum acid phos-

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OSCAR BODANSKY

phate of 3.1 Bodansky units. The patient refused operation and by August had developed osteoblastic metastases. Bilateral orchiectomy was performed, and a bladder biopsy confirmed the presence of prostatic carcinoma. During the next year and a half, he received in succession radiation therapy, estrogen and various types of steroid therapy. Although there was clinical and roentgenographic evidence of the progress of the disease, the serum acid phosphatase never rose above 7.6 units, yet at autopsy there were widespread metastases to practically all tissues. The liver and parts of the bones of the lumbar spine, ribs and sternum were replaced to a considerable degree by large white masses of tumor tissue. The latter case illustrates the dissociation between the extent of metastases and the level of serum acid phosphatase activity. Such a dissociation may be explained by either of two possibilities. First, the acid phosphatase does not pass readily from the metastatic tissue to the circulation. Second, metastatic tissue is an active producer of the enzyme, but the rate of excretion, metabolic degradation, or other mode of disposition of the enzyme may vary greatly with the patient and be excessive in some. With regard to the first possibility, i t is of interest that Nesbit et al. (N2) described a case of prostatic cancer in which extensive metastatic growth recurred in spite of palliative treatment with estrogen. The serum acid phosphatase level remained very low, but histochemical studies revealed an abundance of acid phosphatase in the epithelial cells of the primary and secondary tumors. According to Hertz et al. (H9), autologously transplanted prostatic tissue in dogs became well vascularized, secreted actively, and contained large amounts of acid phosphatase. However, the serum acid phosphatase remained low, even under the stimulus of androgen. Some data are available concerning the metabolic degradation or other type of disposition of acid phosphatase in the circulation. London et al. (L12) studied in some detail the factors involved in the inactivation of serum acid phosphatase. I n vitro experiments showed that the stability of the enzyme was dependent on the temperature, pH, and salt concentration. The rate of inactivation or denaturation a t 37'C was monomolecular; and denaturation rate constants could therefore be derived for serums from normal persons or from various patients with benign hypertrophy or carcinoma of the prostate. Of the several factors found to affect acid phosphatase in vitro, the one that could be tested safely in vivo was alteration of body temperature by application of the procedures of Ripstein et al. (R5). I n two patients

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with carcinoma of the prostate, lowering the body temperature caused a rise in serum acid phosphatase, and, conversely, raising the temperature led to a decrease. In one patient with a benign tumor of the prostate and in two normal individuals, all of whom had normal levels of serum acid phosphatase, lowering the body temperature had no effect on these levels. I n another patient the spontaneous development of fever caused a marked decrease from 585 to 288 Bodansky units. Abatement of the fever was accompanied by a rise in the serum acid phosphatase to 566 Bodansky units (L11). In 1941, Huggins and his associates (H19) had made similar observations on the occurrence of fever in patients with prostatic cancer. On the basis of clinical-biochemical correlations, Hudson e t al. (H16) suggested that the liver may play a role in the metabolism of serum acid phosphatase of prostatic origin. A patient who was diagnosed clinically as having prostatic carcinoma was treated by enucleation prostatectomy, bilateral orchiectomy, and the institution of daily oral diethylstilbestrol. The surgical specimen showed a well differentiated adenocarcinoma of the prostate, but the serum acid phosphatase level was normal and there was no radiographic evidence of metastatic disease. Readmitted five and half years later because of acute urinary retention, there was still no radiological evidence of osteoblastic metastases, and the serum acid phosphatase was still low, 4.2 K.A. units. Several months later the patient was readmitted with a primary complaint of a sudden appearance and progressive enlargement of an epigastric mass. The liver was found to be enlarged, and several liver function tests had now become abnormal. The acid phosphatase had now risen to 201 K.A. units, and before death was 319 K.A. units. Autopsy revealed tumor infiltration to liver, lung, bladder, and lymph glands. The liver architecture was completely destroyed. A second case also showed parallelism between the rise in serum acid phosphatase and a n increase in hepatic damage; whereas a third case demonstrated a parallelism between the fall of acid phosphatase and a return of liver function to normal. 6.6. ACID PHOSPHATASE ACTIVITYIN NONPROSTATIC DISEASE 6.6.1. Introduction The term “serum” has been omitted designedly from the title of this section, for we shall be discussing not only alterations of acid phosphatase activity in the serum of patients with nonprostatic disease, but also in the leukocytes of patients with hematologic and hematopoietic disorders and, in some conditions, in certain specialized tissues.

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OSCAR BODANSKY

6.6.2. Serum Acid Phosphatase Activity in Skeletal Disease

That occasional rises of serum acid phosphatase activity may occur in nonprostatic skeletal disease which may be associated with high serum alkaline phosphatase activity was first observed by Gutman and Gutman in 1938 (G11) and has since been confirmed by others (Jl, 530). I n a study of 32 cases of Paget’s disease, Gutman et al. (G12) found that six of the most advanced cases with diseases of the skeleton and serum alkaline phosphatase levels over 70 Bodansky units had serum acid phosphatase levels above 3.0 K.A. units, the upper limit of normal. The highest activity recorded was 6.5 units. This compared with an incidence of five patients with elevated serum acid phosphatase activity in more than 200 control patients with diseases other than prostatic carcinoma or Paget’s disease. The possibilities that cryptic prostatic carcinoma may coexist with other diseases or that a high serum alkaline phosphatase activity possesses some residual activity a t pH 5.0 tend to be negated by an analysis of these five patients. One female with carcinoma of the breast and osteolytic metastases of the femur, pelvis, and spine had an eIevated serum acid phosphatase activity of 4.2 K.A. units, and a second female with an unknown primary but with osteolytic lesions of the ribs and scapula had an activity of 4.1 K.A. units. The serum alkaline phosphatase activities were 17.8 Bodansky units in the first case and 5.2 Bodansky units in the second case, both above 4.2 Bodansky units, the upper limit of normal values by this method. One female and one male patient had hyperparathyroidism with elevated serum alkaline phosphatase activities and extensive bone changes characteristic of generalized osteitis fibrosa cystica. In both instances, the serum acid phosphatase activity of the serum fell to normal values after removal of the parathyroid adenoma despite transitorily increased serum alkaline phosphatase activity. The fifth patient was a female with osteopetrosis involving the major part of the skeleton. The serum acid phosphatase was 8.7 K.A. units, the highest in the control series-yet the serum alkaline phosphatase was within normal limits. It would appear, therefore, that some patients with skeletal disease may have a slight but definitely elevated serum acid phosphatase activity, at least as determined by the Gutman method (G10, G14), which cannot be explained by concurrent prostatic carcinoma or by a spillover of alkaline phosphatase activity to a p H of 5.0. Table 10 shows the distribution of serum acid phosphatase activities in neoplastic disease other than prostatic cancer. The incidences of elevations were: 19% in patients with skeletal metastases; 2% in pa-

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ACID PHOSPHATASE

TABLE 10 PERCENTILE DISTRIBUTION OF SERUM ACIDPHOSPHATASE IN NORMAL SUBJECTS AND IN PATIENTS WITH DISEASES OTHERTHANOF THE PROSTATE GLANDS Percentage of cases with serum acid phosphatase

Condition 1. Normal subjects 2. Neoplasia other than prostatic carcinoma a. With skeletal metastases b. With liver involvement c. No bone or liver involvement d. Primary bone tumors 3. Nonneoplastic disease of bone a. Paget’s disease b. Hyperparathyroidism c. Miscellaneous diseases of bone

Number of patients

Less than 3.0 K.A. units

2.04.9 K.A. units

5.0-9.9 K.A. unih

6 1

30 240

100

99 46 64

81 98 94

13 2 5

31

90

10

96 9 72

79 67 96

18 11 3

3 22

1

Based on data of Sullivan el al. (530).

tients with liver involvement; 6% in those without either bone or liver involvement; 10% in patients with primary bone tumors. In the category of nonneoplastic diseases of the bone, elevations were present in 21% of 96 patients with Paget’s disease, in 337% of 9 patients with hyperparathyroidism, and in 4% of patients with miscellaneous diseases of the bone. Using the Bodansky (B18, 52) procedure with P-glycerophosphate as substrate, Woodard (W8) was unable to obtain such elevations. She determined the serum acid phosphatase activities in 83 females and 342 males, or a total of 425 patients with miscellaneous diseases. Of these, 61 had various types of infectious or metabolic disorders, including 11 cases of inflammatory disease of bone and 12 cases of hepatic cirrhosis. The remainder had some type of neoplastic disease and about one-third had metastases to bone from cancer of various primary sites. There were 15 cases of osteogenic sarcoma and 32 cases of osteitis deformans. All these cases, whether their serum alkaline phosphatase activities were elevated or not, had serum acid phosphatase values that were essentially within the normal range, O.OW.89 Bodansky unit for females and 0.110.88 unit for males. In contrast to the Gutman method (G10, G14), there-

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OSCAR BODANSKY

fore, the Bodansky method failed to show any elevations, however slight and infrequent, in nonprostatic neoplastic and other skeletal disease. 6.6.3. Copper-Resistant Serum Acid Phosphatase in Miscellaneous Diseases

Abul-Fad1 and King (A4) had reported that a final concentration of 0.0002M Cu2+in the reaction mixture inhibited the hydrolysis of phenyl phosphate by human erythrocytic acid phosphatase to the extent of 859576, but exerted only a slight inhibition, approximately 8%, on the action by prostatic tissue. Aqueous extracts of liver, kidney, and various tumors were inhibited to the extent of 2&30%. Utiliaing these findings and employing the Gutman method (G10, G14), Reynolds et al. (R4) studied the copper-resistant acid phosphatase levels in various miscellaneous diseases. The use of cupric ion was designed to minimize or even eliminate any serum acid phosphatase activity that had originated from red cells. The units of activity were expressed as the number of micromoles of phenol liberated from phenyl phosphate in 1 hour by 100 ml of serum. The serum acid phosphatase activity in 65 healthy volunteers was 11.3 2 5.5 units. The upper limit of normal was 22.3 units. I n a group of 104 female patients with acute or chronic inflammations, arthritis, osteoporosis, hypertension, and/or arteriosclerotic disease, diabetes mellitus, endocrine disorders, hepatic insufficiency or cirrhosis, renal disease, the activities ranged from 0 to 44.0 units, and 14 or 14% had elevated serum acid phosphatase activities, that is, values above 22.3 units. Twenty-one of 105, or 2076, of male patients with these miscellaneous conditions, had elevated serum acid phosphatase levels. This group included 13 patients with prostatic hypertrophy of whom 7, or about 50%, showed elevated activities. Of considerable interest were the much higher incidences of elevated acid phosphatase activities in patients with nonprostatic, metastatic neoplastic disease. For example, in 70 female patients with metastatic carcinoma of the breast, the range of activities was 7.3 to 101 units, with 7476 of the patients showing elevated activities. I n 48 males with nonprostatic metastatic carcinoma and in 42 females with nonmammary metastatic carcinoma, the incidence of elevated acid phosphatase activities were 46% and 31%, respectively. The report of these high incidences of elevations of acid phosphatase activity in miscellaneous disease and in nonprostatic neoplastic disease, determined by a method presumably more specific than the usual Gutman method (G10, G14), is not in accord with earlier studies, such as those of Sullivan et al. (530) shown in Table 10. The incidences of elevations were considerably lower than those reported by Reynolds et al.

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119

(R4).It might have been expected that, with the presumably more specific method proposed by these investigators, the reverse situation would have held. There appear to have been no further clinical studies utilizing the copper-resistant acid phosphatase method. 6.7. SERUMAND PLASMA ACIDPHOSPHATASE ACTIVITYIN HEMATOLOGIC AND HEMATOPOIETIC DISEASE 6.7.1. Thrombocytopenia The development of platelets or thrombocytes takes place chiefly in the bone marrow from primitive totipotential reticulum or stem cells. The normal concentration of thrombocytes in the peripheral circulation of the adult is approximately 250,000-355,000/mm3. Thrombocyte activity is necessary in the process of coagulation. Quantitative platelet deficiency or thrombocytopenia is one of the most common causes of hemorrhagic diathesis, and it may be due either to decreased platelet production or to increased platelet destruction. Any of several basic disorders may account for decreased production. These may include congenital disorders such as hypoplastic anemia, hypoplastic thrombocytopenia, acquired conditions such as nutritional deficiency, toxic depression of the bone marrow, or the replacement of the bone marrow as in leukemia, carcinoma, granuloma, or fibrosis. Increased platelet destruction may include congenital disorders or such acquired disorders as chronic infections, portal hypertension, lymphomas, or thrombotic thrombocytopenia. The presence of acid phosphatase in human platelets was first reported by Alexander (A6) in 1953. Shortly thereafter other investigators confirmed this finding (Pl, S1) . Using P-glycerophosphate as substrate, Zucker and Borelli ( Z l ) determined the acid phosphatase activity directly in platelets separated from human blood; these were washed three times with saline, frozen and suspended in saline in a concentration between 0.135 and 4.2 X lo6 platelets per cubic millimeter. The activity, presumably calculated for 100 ml of a suspension containing 0.43 X lo6 platelets per cubic millimeter, was 0.15 to 0.78 Bodansky units, as compared with a normal serum acid phosphatase activity of 0.0-0.8 Bodansky units. Pedrazzini and Salvidio ( P l ) also determined the acid phosphatase activity directly in platelets, expressing it as micrograms of P liberated in 1 hour from sodium P-glycerophosphate at pH 5.0 by lo1” platelets. The average value from 25 clinically normal individuals was 282 pg of P per hour. I n a subsequent study, Zucker and Woodard (22) prepared what they termed “platelet-poor plasma” by collecting the blood sample in silicone-

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treated glassware, chilling half in ice water, and centrifuging a t 4°C and 20,OOOg for 10 minutes. This sample was substantially free of platelets and other formed elements, and hence of acid phosphatase from these elements. On removal to room temperature, i t underwent some clotting, and the resulting supernatant was termed serum from “platelet-poor plasma.” The remaining half of the sample collected in the silicone-treated glassware was transferred to ordinary glass and allowed to clot a t room temperature for 30 minutes, as is usually done to obtain serum for acid phosphatase determinations. The mean values and their standard deviations for serums from “platelet-poor plasma” from various groups of subjects were as follows in Bodansky units: 28 normal women, 0.094 k 0.009; 23 normal men, 0.109 k 0.021. The corresponding values for ordinary serum acid phosphatase were 0.226 k 0.13 and 0.278 ? 0.27. It appeared, therefore, that approximately 60% of the acid phosphatase in serum arises from the liberation of this enzyme from platelets as a result of clotting. The moiety of serum acid phosphatase activity that may be derived from platelets has also been evaluated by Oski et al. (021, employing the Gutman method (G10, G14). These investigators avoided the use of the vacuum tube (Vacutainer) and withdrew blood by syringe and gently ejected it into a glass tube containing ammonium oxalate-potassium oxalate as anticoagulant. The blood was immediately centrifuged at 4°C for 15 minutes at 7009; the supernatant plasma was removed without disturbing the buffy coat, transferred to a plastic tube, and recentrifuged a t 4°C for 30 minutes a t 40008. The plasma was aspirated and found to be free of platelets both by direct platelet counting and by examination of a Wright-stained smear of a centrifuged aliquot. The mean values and the standard deviations for this platelet-free plasma acid phosphatase were determined for groups of children at various ages and for adults. Each group consisted of 10 or 11 individuals. The values, expressed as K.A. units, were as follows: 1.0-3.0 year olds, 4.5 k 1.0; 3.1-6.0 year olds, 4.2 k 0.7; 6.1-9.0 year olds, 4.1 k 0.8; 9.116.0 year olds, 3.9 f 0.9 K.A. units. The value for male adults were 2.3 f 0.3 K.A. units and that for female adults, 2.2 f 0.3. The mean plasma acid phosphatase activities declined with increasing age. Although the activity of plasma acid phosphatase was less than the corresponding serum activity, these differences were not large, ranging from 0.1 to 1.9 units. Reports on the alteration of serum and/or plasma acid phosphatase activity in thrombocytopenia have not always been consistent. Zucker and Woodard (22) described a series of 12 patients with thrombocyto-

ACID PHOSPHATASE

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penia secondary to a variety of conditions such as carcinoma of the breast, acute and chronic leukemias with platelet counts chiefly between 2000 and 60,000. The mean value for the acid phosphatase activity in serum from platelet-poor plasma was essentially normal, but the value for the activity in ordinary serum was 0.123 Bodansky unit, significantly less than the values of 0.226 unit for normal women and 0.278 unit for normal men. These findings indicated again that the platelets were a major source of the acid phosphatase liberated into serum during clotting (22). Oski et a2. ( 0 2 ) classified their cases of thrombocytopenia as arising from impaired production of platelets, from increased destruction of platelets, or from a combination of these factors. In a group of 8 children with bone marrow failure, evidenced by low platelet counts and megakaryocytic hypoplasia or aplasia without complicating infection or drug therapy, each patient had a plasma acid phosphatase activity lower than the mean value for its age. The differences were, however, not large. For example, in a child with acute leukemia aged 2 years and 7 months, the platelet count was 30,000 and the plasma acid phosphatase was 3.6 K.A. units, as compared with a value of 4.5 k 1.0 K.A. units for normal children in this age group. The comparison of the plasma acid phosphatases in this group of 8 children, as a whole, with those in the normal group yielded a significant p value of less than 0.01. In general, therefore, these findings are in agreement with those of Zucker and Woodard (22) for patients with thrombocytopenia due to bone marrow failure. In a group of six children with acute thrombocytopenia and bone marrow megakaryocytic hyperplasia, the blood plasma, prepared as previously described, showed, in each case, an acid phosphatase activity, as determined by Gutman’s method, that was higher than the mean value for that age ( p = 0.02). The p value for the comparison of the group as a whole with normals was between 0.01 and 0.02. I n all six of these patients the plasma acid phosphatase values returned t o normal or near normal levels as the thrombocytopenia was corrected. Oski e t a2. ( 0 2 ) also studied 15 cases of chronic idiopathic thrombocytopenic purpura in whom the bone marrow showed normal to increased numbers of megakaryocytes. Of these, 13 showed plasma acid phosphatase values that were elevated above the normal mean for their age, albeit some of these differences were small. However, these elevations were statistically significant with a p value less than 0.01. I n essence, then, these investigators felt that the plasma acid phosphatase activity, unobscured by in vitro destruction of platelets, could reflect the contribution of acid phosphatase from in vivo platelet destruction in various types of thrombocytopenias. To summarize, in thrombocyto-

122

OSCAR BODANSKY

penias with a deficiency of megakaryocytes in the bone marrow, the plasma acid phosphataee activity was significantly lower than that in normal controls, whereas it was higher than normal in patients with chronic or idiopathic thrombocytopenic purpuras and normal or increased numbers of megakaryocytes in their bone marrow. However, Cooley and Cohen (C9) studied nine cases of idiopathic thrombocytopenic purpura in which they failed to find any consistent correlation between the plasma acid phosphatase activities and platelet counts. Cohen et al. (C6) had shown that this condition could be classified into two major types, destructive and nondestructive, comprising 80% and 20%, respectively, of the total. I n sequential studies of their various patients, Cooley and Cohen (C9) did not find any increased plasma acid phosphatase activity in those showing the destructive type, although in two cases of nondestructive (hypoplastic) thrombocytopenias, the plasma acid phosphatase activities were usually normal or low. Cooley and Cohen (C9) also found that in a group of eight patients with secondary (nondestructive) thrombocytopenias with nearly normal platelet life-spans (mostly 5-7 days) the plasma acid phosphatase levels tended to be low and to be correlated with the platelet count. Pedrazzini and Salvidio (Pl) found that the average value of acid phosphatase activity in platelets from 25 patients with hypoprothrombinemia secondary to Morgan-Laennec’s hepatic cirrhosis was 119 pg of P per hour per 1Olo platelets, significantly less than the normal value of 282 pg of P. These investigators also found that ATPase, 5’-nucleotidase, and alkaline phosphatase activities were reduced in the platelets of the patients with liver cirrhosis by 44%, 58%, and 67%, respectively. Along with the reduction in acid phosphatase, these changes were considered to be an expression of functional damage to the platelets. 6.7.2. Serum and Plasma Acid Phosphatase in Thrombocytosis The presence of greater than normal amounts of thrombocytes in the circulation is known as thrombocytosis and along with reticulocytosis and leukocytosis is a manifestation of increased activity of the hematopoietic system. Zucker and Woodard (22) reported a series of 12 patients with thrombocytosis, consisting of two cases of polycythemia Vera, three of essential thrombocytemia, three of chronic granulocytic leukemia, one myeloproliferative syndrome, one erythroleukemia, and one cancer of the bladder. The platelet counts ranged from 685 x los to 2500 x lo3 per cubic millimeter, all much above the upper limit of normal. The serum acid phosphatase activity was determined by the Bodansky method (B18,52) with P-glycerophosphate as substrate. The mean value for the series of 12 patients was 0.983 -+ 0.122 Bodansky unit, consider-

ACID PHOSPHATASE

123

ably elevated above the normal mean values of 0.226 2 0.0126 unit for women and of 0.278 2 0.0270 unit for men. This elevation represented the release on clotting of acid phosphatase from the large number of platelets. The mean value for acid phosphatase activity of platelet-poor plasma in the series of these 12 patients with thrombocytosis was 0.141 2 0.0162 unit and was not significantly different from the mean values of 0.094 2 0.0091 unit for normal women, 0.109 2 0.021 unit for normal males or of 0.134 2 0.018 unit for patients with thrombocytopenia. Accordingly, it may be concluded that in thrombocytosis there is little in vivo contribution by the platelets to the circulating acid phosphatase. 6.7.3, Serum Acid Phosphatase Activity in Other Myeloproliferative Disease The term “myeloproliferative disorders” has been applied to all those conditions which are characterized by proliferation of cells in bone marrow or in other sites of extramedullary blood formation. The overgrowth is self-perpetuating and involves one or more lines of bone marrow elements (myelocytic, erythrocytic, megakaryocytic) and cells like fibroblasts derived from the reticulum. We have already mentioned some of these conditions, for example, chronic granulocytic leukemia and myeloid metaplasia, in connection with our discussion on the relationship between serum acid phosphatase and the platelet count. Employing /3-glycerophosphate as substrate in the Bodansky method, Bases (B6) found that 9 of 16 patients had slight, but significant, elevations of serum acid phosphatase activity-in one case as high as 4.7 Bodansky units. In general, a proportionality existed between the enzyme activity and the platelet or white blood cell count. In sequential studies of individual patients, particularly in connection with a chemotherapeutic regimen, the change in phosphatase activity paralleled both the white cell and platelet counts, but occasionally the parallelism existed only between the enzyme activity and the white cell count. I n this connection it may be recalled that, according to Zucker and Borelli ( Z l ) , the clotting of blood involves the destruction of platelets and possible release of their acid phosphatase. Valentine and Beck ( V l ) demonstrated that white cells from patients with chronic granulocytic leukemia in general had abnormally high levels of acid phosphatase. It would appear, therefore, that in Bases’ cases of myeloproliferative disease, both the platelets and the granulocytes might have been the source of the acid phosphatase elevations (B6). Bases (B6) found no elevations of the serum acid phosphatase activity in a group of 20 patients with chronic lymphocytic leukemia, other lymphomas, leukemoid reactions, and acute leukemia. This would ap-

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pear reasonable in view of the findings that leukocytes from patients with acute leukemia or lymphatic leukemia either had normal or low acid phosphatase activity, as determined with P-glycerophosphate as substrate (B10, V l ) . However, Klastersky and Coune (K8) recently reported a case of a male with lymphoblastic leukemia and elevated values of serum acid phosphatase, ranging from about 4 to 8 units; nitrophenyl phosphate was used as the substrate. These elevated values decreased and even reverted to normal values, 0.0 to 0.63 unit, after administration of vincristine. The decrease in enzyme activity paralleled the decrease in the number of blasts in the peripheral blood and percentage in the marrow. During continued treatment for 4 months, the number of blasts, their percentage in the marrow, and the serum acid phosphatase activity continued a t low levels. At the end of this period, the patient became resistant to vincristine, and the blasts and the serum acid phosphatase activity rose. Again, the values for these parameters fell with the institution of methotrexate therapy, but death occurred following massive hematuria. Postmortem examination showed leukemic infiltration of the spleen, bone marrow and laterotracheal nodes, but the prostate was clear of carcinoma or of leukemic filtrate. Although the serum acid phosphatase activity was inhibited by L-( +)-tartrate, and spleen acid phosphatase has also been reported to be inhibited by this compound (A4), it is difficult to ascribe any definite basis for the increased serum acid phosphatase in the case reported by Klastersky and Coune (K8). 6.8. ACIDPHOSPHATASE ACTIVITY IN GAUCHER’S DISEASE 6.8.1. Serum Acid Phosphatase in Gaucher’s Disease

The characteristic lesion in this disease is the infiltration of spleen and, to a lesser extent, of liver, bone marrow, and lymph nodes with Gaucher cells. These cells have small dark central or eccentric nuclei and clear or foamy cytoplasm with fibrillar striations. Gaucher’s disease is one of the lipidoses and is caused by an inherited deficiency of glucocerebrosidase, the enzyme required for the degradation of glucocerebroside (B28). As a result of the phagocytosis of cells and cellular debris, large amounts of glucocerebroside are deposited in the reticuloendothelial cells, causing them to assume the typical wavy, fibrillar appearance of Gaucher’s cells. I n 1957, Tuchman and Swick (T6) reported the finding of elevated serum acid phosphatase activities, ranging from 7.0 to 10.3 K.A. units in a 68-year-old man, who was first suspected of having carcinoma of the prostate. The diagnosis of Gaucher’s disease was then considered and confirmed by a sternal marrow aspiration. By 1959, Tuchman et al. (T7)

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125

had collected a series of 12 patients who showed a range of 7 to 14.3 K.A. units of serum phosphatase activity when determined by the method of Gutman (G10, G14). Tuchman et al. (T7,T8) stated th a t the upper limit of normal in their hospital laboratory was 4 K.A. units and that a range between 4 to 5 K.A. units was equivocal. Neither Cu2+or tartrate reduced any, and formaldehyde reduced only one, of the 12 activities to below 5 K.A. units, thus characterizing this acid phosphatase activity as different from the acid phosphatases of erythrocyte liver, prostate, spleen, or bone marrow. Using P-glycerophosphate as substrate, Tuchman et al. (T7) reassayed the serum acid phosphatase activities in his series of 12 patients with Gaucher’s disease. In contrast to the Gutman method, which had yielded elevated values in all cases, only four of the patients now showed elevations above the upper limits of normal, approximately 0.8 Bodansky unit. The two patients who had shown the highest values, 10.2 and 14.3 K.A. units by the Gutman method (G10, G14) had one normal value, 0.75 Bodansky unit, and one slightly elevated value, 0.88 unit. Nonetheless, elevation of serum acid phosphatase activity in Gaucher’s disease with phenyl phosphate as substrate cannot be considered as nonspecific or spurious. Tyson et a2. (T9) reported the case of a 65-year-old man who was considered to have cirrhosis of the liver because of hepatosplenomegaly, anemia, pancytopenia, and esophageal varices. On admission to the hospital three years later, skeletal survey revealed some areas of lucency in the femurs compatible with Gaucher’s disease, multiple myeloma, or myeloproliferative diseases. The serum acid phosphatase level was consistently elevated, from 12.9 to 13.7 K.A. units and about 2.0 Bodansky units. Sternal marrow biopsy was refused by the patient. After his death five years later, autopsy and microscopic examination revealed infiltration of the liver, spleen, bone marrow, and lymph nodes with typical Gaucher cells. It has already been noted that Gaucher’s disease is characterized by infiltration of the spleen and, to a lesser extent, of the liver, bone marrow, and lymph nodes with the typical Gaucher cells. Confirming earlier observations that these cells stained for acid phosphatase, Crocker and Landing (C10) suggested the possibility that the elevated serum acid phosphatase activity in patients with Gaucher’s disease might reflect a spillage of the enzyme from the spleen and other tissues. Using P-glycerophosphate as a substrate, these investigators obtained average activities of 330 Bodansky units per gram for spleens from 4 children with miscellaneous disease, of 279 units per gram from 4 children with Niemann-Pick’s disease, and a markedly elevated value of 875 units per gram from spleens from 7 children with Gaucher’s

126

OSCAR BODANSKY

disease. Five of the children in this last group, all of whose serum acid phosphatase activities were elevated, as determined by the Gutman method with phenyl phosphate as substrate (G10, G14), were subjected to splenectomy. I n four of these, the serum acid phosphatase activity decreased precipitously by 40-55747. 6.8.2. Isoenzymes of Acid Phosphatase in Serum and Spleen Cell Suspensions of Gaucher’s Disease In 1964, Czitober et al. (C11) reported three patients with Gaucher’s disease in whom the serum acid phosphatase exhibited five zones of activity. Using disc electrophoresis on polyacrylamide-gel columns, as described by Davis (D5), Goldberg and his associates (G6) determined the isoenzyme pattern in the serums of nine patients with Gaucher’s disease who had elevated serum acid phosphatase activities ranging from 8 to 25.8 K.A. units. In the group as a whole, five bands of acid phosphatase activity were discernible. The most anionic migrated in a position similar to transferrin and was designated as band I. Band I1 was in the fast moving haptoglobulin region, and bands 111, IV, and V had mobilities of y-globulins and the slower haptoglobulins. All nine patients had a t least two bands, I and 11, of which the latter was the broadest and most intensely stained. One or more of the minor bands, 111-V, were considerably narrower and were present in five of the nine patients. Of these five patients, three had three bands and two patients had two minor bands. One patient showed all five bands. Li e t al. (L8) studied spleen cell suspensions, with and without cotton filtration, from a patient with Gaucher’s disease. The preparation, without filtration, showed a strong isoenzyme 1 and a new isoenzyme, which remained at the cathode. This isoenzyme was distinctive, not being found in leukocytes, and was designated as isoenzyme No. 0. It should be noted that Li et al. (L7, L8) designated the isoenzymes as ranging from No. 1, cathodic, to No. 5 , the most anionic. After removal of the Gaucher cells from the spleen cell preparation by cotton filtration, the remaining leukocytes did not show isoenzyme No. 0, but isoenzymes Nos. 1, 2, 3, and 4 were now apparent. Isoenzyme No. 0 was therefore characteristic of the Gaucher cell. 6.9. LEUKOCYTIC ACID PHOSPHATASE ACTIVITY IN HEMATOLOGIC AND

HEMATOPOIETIC DISEASE

6.9.1. Leukocytic Acid Phosphatase in Normals The acid phosphatase activity of leukocytes was determined by Valentine and Beck (Vl) in 1951 and expressed as the amount of phos-

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127

phorus liberated from 0.02 M sodium p-glycerophosphate in 1 hour a t 37°C by 1O1O cells. The reaction mixture was buffered with acetateVerona1 a t pH 5.0 and contained 1 mg per milliliter of a solution of saponin to lyse the cells. The range in a series of 23 controls was 16-37 mg, and the mean was 22 mg. Using the method of Allen and Gockerman (AT), Li et aE. (L7) recently found the average for the total acid phosphatase activity of the leukocytes from five normal persons to be 1219 nmoles of naphthol liberated from 0.005 M sodium a-naphthyl acid phosphate per hour per lo7 cells at p H 5.0 and 25°C. Electrophoresis was carried out by Li et al. (L7) by the method of Axline (A18). Optimal separation was obtained a t p H 4.0 on a 7.5% acrylamide gel matrix containing 0.5% Triton X-100. Electrophoresis was carried out a t 4°C for 50 minutes with a current of 4 mA/tube. Four isoenzymes of acid phosphatase were obtained for normal leukocytes, proceeding from the cathodic, No. 1, to the most anionic, No. 4. The mean normal values for the distribution of activity among these isoenzymes were No. 1, 37.8%; No. 2, 29.2%; No. 3, 11.5%; No. 4, 21.5%. I n a subsequent study, Li and his associates (La) found th a t preparations of 10 X lo6 lymphocytes or of 10 X 10' platelets per milliliter of 5% Triton X-100 each gave, upon electrophoresis, only one band of isoenzyme activity, No. 3. It was the neutrophiles and monocytes that contributed isoenzymes 1, 2, and 4. I n normal cases the neutrophiles were as high as 70-800/0. The acid phosphatase activities of these bands in neutrophiles or monocytes were sufficiently high so that even as little as 5% of either cell type in the blood of patients with chronic lymphocytic leukemia yielded clear evidence of isoenzymes 1, 2, and 4.

6.9.2. Leukocytic Acid Phosphatase i n Leukemia I n 1951, Valentine and Beck (B8, V l ) found that, in spite of considerable interindividual variability, leukocytic acid phosphatase was elevated in chronic granulocytic leukemia and tended to be decreased in chronic lymphocytic leukemia and in acute leukemia (Table 11). Statistical treatment of their original data shows that only the mean values for alterations in chronic granulocytic (myelocytic) leukemia and acute leukemia were significantly different ( p < 0.01) from the mean value for leukocytic acid phosphatase in normals. Using the method of Allen and Gockerman (A7) and a-naphthyl phosphate as substrate, Li et al. (L7) found, in agreement with Valentine and Beck ( V l ) , that the total leukocytic acid phosphatase activity was decreased in acute granulocytic and lymphocytic leukemias. However, in contrast to the findings of Valentine and Beck ( V l ) , Li et al. (L7) observed no elevation in

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TABLE 11 ACIDPROSPHATASE IN THE LEUKOCYTES OF NORMAL SUBJECTS AND PATIENTS WITH LEUKEMIA’

Group

No. of subjects

No. of determinations

Range

Mean

23 30 14

23 30 22

14-37 7-66 16-61

22 26 35

12

16

1-106

18

8

14

0-46

9

_ _ _ _ _ _ _ _ _ ~

Normals Leukocytosis Chronic granulocytic leukemia Chronic lymphocytic leukemia Acute leukemia ~

~

Data of Valentine and Beck (VI) and of Beck and Valentine (B8). Activities are expressed aa milligrams of phosphorus liberated in 1 hour by 1010 cells from a reaction mixture at pH 5.0, containing a final concentrationof 0.02 M sodium 8-glycerophosphate aa substrate and 1 mg per milliliter of saponin to lyse the leukocytes.

the mean value for chronic granulocytic leukemia (Table 12). It was also of interest that the total activity was greatly reduced in chronic lymphocytic leukemia. The distribution of the four normal isoenzymes in various types of leukemia is also shown. Electrophoresis was carried out on polyacrylamide gels by the method of Barka (B3) ; the substrate was sodium a-naphthyl acid phosphate, and the diazonium salt of O-aminoazotoluene (fast garnet GBC) was the coupler. The relative intensity of each phosphatase band in the gels was estimated with a Gilford densitometer. The shift in isoenzyme pattern from the normal may be readily appreciated (Table 12). Thus, in acute granulocytic and chronic lymphocytic leukemias, the fraction of isoenzyme 3 was increased, whereas that of isoenzyme 2 was decreased substantially. In addition to the four bands of acid phosphatase isoenzyme activity in normal leukocytes, two other isoenzymes may appear in leukemia. In a case of acute granulocytic leukemia with 100% blast forms, only one electrophoretic band of activity was manifest; this migrated between normal band 3 and 4 and was therefore designated 3b. In a case of leukemic reticuloendotheliosis with 98% reticulum cells, only one, and this a strongly staining band, was present; this migrated anodically beyond No. 4 and was therefore designated No. 5 (L8).

6.9.3. Leukocytic Acid Phosphatase in Leukemic Reticuloendotheliosis This condition-which has also been known under the names of “reticulum cell leukemia,” “lymphoid reticular cell neoplasia,” and “hairy cell disease” (M8)-is characterized by massive splenomegaly

TABLE 12

LEUKOCYTE ACID PHOSPHATASE ACTIVITYIN VARIOUSHEMATOLOGIC DISEASES~ ~~

Fractions Disease Normal

5

Chronic granulocytic leukemia

4

Acute granulocytic leukemia

3

Acute lymphocytic leukemia Chronic lymphocytic leukemia ~

~~~~

1219 (926-1650)” la42 (826-1700) 649 (120-1300) 826

1 7

284

(33-836) ~

Based on data of Li d al. (L7). b Expressed as nanomoles of naphthol per hour per 107 cells. c Detected in one case. d Numbers within parentheses indicate range. a

37.8 (2848.8) 35.1 (23.7-48.3) 35.2 (17.5-53.1) 29.2 29.9 (049.7)

29.2 (25-36.7) 32.6 (27.5-38.6) 12.7 (10.9-14.5) 39.7 14.7 (0-29)

11.5 (8.2-15.7) 7.3 (5.0-9.7;) 29.4 (15.647.4) 8.0 42.5 (13.6-100)

21.5 (17.3-26) 25 (17.5-32.7) 22.7 (18.8-25.1) 23.1 12.9 (0-23.9)

Trace -

-

W

; 0v

E

e

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OSCAR BODANSKY

due to invasion of reticulum cells. The peripheral blood and bone marrow contain large numbers of a typical large “lymphoid reticular cell,” 1220 p in diameter, with a round or oval, occasionally kidney-shaped, eccentrically placed nucleus and a plentiful, very faintly basophilic and cloudy cytoplasm (L5). Ten of a series of 25 patients, recently reported by Lee et al. (L5), had moderate anemia, and all but 3 had varying degrees of thrombocytopenia. At the first visit, 14 of the 25 patients had reduced total white blood cell counts, less than 5000 per cubic millimeter. I n all patients, the differential white cell count was very abnormal. As was noted above, a preparation obtained from patients’ peripheral blood in which 98% of the leukocytes were reticulum cells, showed only one isoenzyme, No. 5, of acid phosphatase activity (L8). I n patients with a differential white cell count with lesser numbers of reticulum cells and greater numbers of neutrophiles and lymphocytes, isoenzymes 1, 2, 3, and 4 were also evident. For example, in a case with 54% reticulum cells, 20% neutrophiles, 1% monocytes, and 25% lymphocytes, the relative isoenzyme activities were: No. 0, 0% ; No. 1, 30.8%; No. 2, 18.8%;No. 3, 9.776; No. 3B, 8.0%; No. 4, 10.8%; No. 5, 21.9% (L8)* L-( +)-Tartrate (0.05 M ) inhibited isoenzymes Nos. 1 4 but had no appreciable effect on the reticular cell isoenzyme, No. 5 (M8, Y l ) . I n cytochemical studies of blood smears from three patients with leukemic reticuloendotheliosis, the acid phosphatase activity in the monocytes, eosinophiles, neutrophiles, and other cells that could definitely be identified as lymphocytes did not differ appreciably from those of normal subjects. In all three patients the neoplastic reticulum cell showed various degrees of acid phosphatase activity; most of them were strongly positive. The enzyme activity in these cells was resistant to to L-( + ) -tartrate, whereas it was completely inhibited in other types of cells. 6.9.4. Leukocytic Acid Phosphatase in Other Hematologic Disease Li et aZ. (L7) have also studied the leukocytic acid phosphatase activity and the distribution of its isoenzymes in other hematologic diseases. The average values for the total activity and the distribution of the four isoenzymes in three cases of hemochromatosis were not significantly different from those observed in normals (Table 11). This also held for the averages and ranges for three cases of polycythemia Vera. However, the average value, 5.776, and the range, 4.5-7.376, for isoenzyme 3 in three cases of Hodgkin’s disease appeared lower than the corresponding values, 11.5% with a range of 8.2-15.7% for the five normal individuals (Table 12). Three cases of infectious mononucleosis had

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131

an average activity of 721 (424-1000) nmoles of naphthol liberated per hour per lo7 cells, which was distinctly lower than the normal value. The distribution of activity for the four isoenzymes was: No. 1, 51.4% ; No. 2, 12.8%; No. 3, 19.5% and 16.3%, with isoenzyme 1 activity appearing distinctly higher, and isoenzyrne 2 activity distinctly lower, than the corresponding values for normals (Table 12). 6.10. SERUM ACIDPHOSPHATASE I N THROMBOEMBOLISM Schoenfeld et al. (57, S8, S9) have reported the occurrence of small but definite elevations of serum acid phosphatase activity, as determined by the Gutman method (G10, G14), in myocardial infarction, pulmonary embolism, peripheral thromboembolism and arterial embolism. The following possible explanations may be invoked for this phenomenon: (a) degeneration of enzyme-rich parenchymal tissue subserved by the occluded vessel ; (b) autolysis of enzyme-rich cells, including platelets and erythrocytes, enmeshed in the blood clot ; (c) generalized hypoxic injury to, and release of acid phosphatase from, various organs; (d) thrombocytosis caused by stress-induced splenic contraction and by increased thrombocytogenesis due to tissue necrosis. However, not sufficient evidence was available to decide among these alternatives, or to assess the contributions of each to the elevated serum acid phosphatase level. 6.11. SERUMACID PHOSPHATASE IN DISEASESOF CHILDHOOD Although serum acid phosphatase activity has already been considered in certain diseases of childhood, such as leukemia or Gaucher’s disease, i t may be of value to make several general comments in this area. Laron and Kowadlo (L4) found that the mean normal values for total serum acid phosphatase were: 5.23 k 1.26 K.A. units for children 1 year of age and 4.63 f 0.93 K.A. units for children 2-8 years of age. These are higher than the values for adults. The mean value for L-( + ) tartrate-inhibited acid phosphatase activity was 0.21 k 0.28 K.A. units for all age groups. Of 24 children with rheumatic fever, 6, or 25%, had total serum acid phosphatase activities above 6.5 K.A. units or the mean normal value, 4.63 K.A. units plus 2 standard deviations. In 2 of 9 infants with rickets, the total serum acid phosphatase activity was abnormally high, 11.3 and 9.7 K.A. units. High values were also obtained in occasional cases of nephrosis, pneumonia, and hepatitis. Of 23 children with rheumatic fever in which tartrate-inhibited serum acid phosphatase activity was determined, 12 had values above 0.77 K.A. unit, or the mean value, 0.21 K.A. unit plus two standard deviations.

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7.

lysosornal Disease and Acid Phosphatase Activity

7.1. INTRODUCTION During the past several years a number of reports have appeared indicating that lysosomes and the acid hydrolases characteristic of them may play a role in several types of human disease. Weissmann (W3) has suggested that lysosomes may be an important factor in autoimmune phenomena and connective tissue diseases, such as systemic lupus erythematosus, rheumatic fever and polymyositis. According to this thesis, degradative enzymes, released from lysosomes, may denature the native constituents of cells or connective tissue. Such denatured products could then induce the formation of circulating antibodies, which would be directed not only against denatured constituents but also against antigenically related normal tissues. Gordis (G8) has further reviewed the pathological effects of lysosomal abnormalities, such as deficiency or excess of hydrolases, the stability of lysosomes, and the possible types of disease each of these effects would lead to. 7.2. LYSOSOMES AND CANCER The selective concentration of hydrocarbon carcinogens in lysosomes and the production of chromosomal damage following lysosomal disruption have been reported by Allison and his associates (A8, A9). Scherstbn et al. (S5, S6) have shown that both the free and total activities of the lysosomal enzymes-acid phosphatase, aryl sulfatase, cathepsin, and p-glucuronidase-in the livers of patients with malignant renal tumors were significantly elevated. In kidney tissue without tumor growth, these lysosomal enzymes were of the same order of magnitude as in normal tissues. The enzyme activities in renal carcinoma tissue were lower than in liver and in kidney tissue. Schersten e t al. (S6) suggested that lysosomal enzymes may be released from tumor tissue and then taken up by other tissues. 7.3. DEFICIENCY OF LYSOSOMAL ACID PHOSPHATASE In 1970, Nadler and Eagan ( N l ) described a new familial metabolic disorder which, quite accidentally, was found to be characterized by a deficiency of lysosomal acid phosphatase activity. This finding was observed in the male child resulting from the fourth pregnancy of a 34-year-old Puerto Rican woman. The previous three pregnancies had resulted in the birth of children who had survived for periods ranging from 2 hours to 11 months. The full-term male infant resulting from the fourth pregnancy behaved normally a t first, but developed lethargy

133

ACID PHOSPHATASE

and incidents of vomiting at about the age of 3 months and was admitted a t this time because of these symptoms and fever. The liver was enlarged to 4 cm below the right costal margin. He died several days after admission. I n the course of studies of the nuclear, mitochondrial, lysosomal, microsomal, and supernatant fractions of the homogenates of fibroblasts grown from skin biopsies, it was found that the activities of glucose-6phosphate dehydrogenase, lactic dehydrogenase, a-glucosidase, and P-glucuronidase were normal. In contrast, the activity of acid phosphatase was reduced in all fractions and was virtually absent in the lysosomal fraction. Addition of Triton did not appreciably increase the lysosomal acid phosphatase activity. Table 13 shows that the acid phosphatase activities in the lysosomal fraction from the two obligate heterozygotes (the parents, who were first cousins) and in five other members of the family (presumably heterozygotes) were approximately 50% of the activities in the control family. The mode of inheritance was therefore apparently that of an autosomal recessive defect. The acid phosphatase activities were also determined in brain, kidney, liver, and spleen taken at autopsy from the patient and, like the acid phosphatase activities of lymphocytes and the fibroblasts, was found to be virtually absent, certainly less than about '2% of the activities in the corresponding tissues of the control individuals. Lymphocytes obtained from whole blood showed essentially the same acid phosphatase activity in the heterozygotes as in the controls. However, after 56 hours of stimulation with phytohemagglutinin (PHA) , the acid TABLE 13 ACIDPHOSPHATASE ACTIVITYOF CULTIVATED FIBROBLASTS IN LYSOSOMAL ACID PHOSPHATASE DEFICIENCY~

Subjects

Original homogenate

Lysosomal fraction

Lysosomal fraction Triton

Controls (15)b Obligate heterozygotes (2) Presumable heterozygotes ( 5 ) Patient (1) Abortus (1)c

6.2 f 0.5d 2.8 f 0.2 2.7 f 0.3 1.2 f 0.1 1.0 f 0.1

7.2 f 0.7 3 . 0 f 0.9 3.3 f 1.1 0.1 f 0 . 1 0.2 f 0 . 1

9.6 4.2 4.3 0.2 0.2

~~~~~~

~

~

~

+

1.9 1.3 1.5 0.1 0.1

f f f f f ~

Based on data of Nadler and Eagan (Nl). * Figures in parentheses indicate the number of patients studied. e Detected in utero by examination of amniotic fluid cells at week 13 of pregnancy. d Values are expressed as micromoles of p-nitrophenol formed per hour per microgram of protein. a

134

OSCAR BODANSKY

phosphatase activity of the lymphocytes in the heterozygotes showed about one-third the activity of that in the controls. The report by Nadler and Eagan (Nl) raises certain general questions about the metabolic role of acid phosphatase. It is a t present difficult to determine how intermittent vomiting, hypotomia, lethargy, opisthotonus, and terminal bleeding may be related to the absence of lysosomal acid phosphatase. Yet a few gleanings from the literature indicate that some general cellular metabdlic role for acid phosphatase may exist. As was previously noted, DiPietro and Zengerle (D13) were able to separate and purify three isoenzymes of acid phosphatase from the human placenta. Isoenzyme I11 possessed properties of potential metabolic interest in that it was activated to a considerable extent by the purines, adenine, and 6-ethylmercaptopurine and was inhibited by pyridoxine 5-phosphate (vitamin B,) . However, these activities were not observed with organic phosphate substrates other than p-nitrophenyl phosphate. It has also been suggested that acid phosphatase may participate in the regulation of pyridoxal phosphate-requiring enzymes (A10). 7.4. MULTIPLELYSOSOMAL ENZYME DEFICIENCY

Within the past few years, several patients have been described who have a hereditary defect characterized by severe psychomotor retardation, shortness of stature, intermittent respiratory infections, and slowly progressive “Hurler-like” changes of facies and bony configuration. Fibroblasts grown from a skin biopsy have inclusion bodies and, hence, have been considered to display the “I (for inclusion) cell phenomenon.” The disease has also been referred to as “I-cell disease.” A number of lysosomal enzymes in the fibroblasts of the patient have been found to have greatly reduced activities. Thus, in a recent study, Wiesmann et al. (W5) reported that arylsulfatase A activity was reduced to 20% of normal ; p-galactosidase, N-acetyl-p-galactosaminidase, N-acetyl-p-glucosaminidase, and P-glucosaminidase activities were as low as 2-1076 of normal, and a-fucosidase was not detectable. Intermediate enzyme activities were found in the fibroblasts of the mother of the patient. Nonlysosomal enzymes, such as malic dehydrogenase and lactic dehydrogenase, showed normal activity but curiously enough, the typically lysosomal enzyme, acid phosphatase, also showed normal activity. Testing of the medium in which the fibroblasts had been growing for 3 days revealed significantly elevated levels of activity for the lysosomal enzymes that had been decreased within the cell. No mention was made of acid phosphatase activity. Wiesmann e t al. (W5) considered several

ACID PHOSPHATASE

135

possible explanations, but concluded the most likely one to be a basic defect in the passage of lysosomal enzymes into the outward medium. However, no explanation was offered for the absence of any change in the acid phosphatase activity. Sawant et al. (S3) have investigated several factors that affect the permeability of the rat liver lysosomal membrane and the rates of outward passage of the various lysosomal enzymes. It is possible that, if lysosomal permeability be a characteristic of “I-cell disease,” such permeability is not general and does not apply t o acid phosphatase. Again, Rahman et al. ( R l ) has reported that rat liver lysosomes may be heterogeneous in terms of their enzyme contents. If such a consideration were to apply to lysosomes from other tissues and species, it is conceivable that lysosomes containing p-galactosidase and the other enzymes which are reduced in activity would be damaged whereas those containing acid phosphatase would be more resistant.

7.5. HEMORRHAGIC ENTEROPATHY AND LYSOSOMAL ENZYMES I n the past few years, there has been an increasing interest in the hemorrhagic enteropathy that may be associated with myocardial infarction, terminal cardiac diseases which involve heart failure, valvular heart disease with or without operation, and open-heart surgery (C2). The damaged intestinal mucosa cells may release their potent hydrolytic lysosomes into the lumen and, indeed, into the blood through the thoracic duct (B13). Chiu et al. (C2) have recently studied the intestinal lesions and circulating lysosomal enzymes in extracorporeal circulation both in experimental and in clinical situations. With respect to the latter, 25 adult patients undergoing cardiopulmonary bypass for open-heart surgery were investigated. Arterial blood samples were obtained prior to and immediately after cardiac bypass, and several hematologic and biochemical parameters, including serum acid phosphatase and ,8-glucuronidase, were determined. Serum acid phosphatase activity was determined by a commercially available method with p-nitrophenyl phosphate as substrate (C2) and p-glucuronidase by the method of Fishman e t al. (F4). There was no significant difference between the serum p-glucuronidase activities before and after the bypass. However, the serum acid phosphatase was higher, in each of the patients but one, after the bypass than before, and the mean value was significantly higher. The basis for the increased serum activity of one lysosomal enzyme, acid phosphatase, but not that of another, P-glucuronidase, is of interest. It is possible, as Chiu et al. (C2) have suggested, that the increased acid phosphatase

136

OSCAR BODANSKY

activity may reflect largely the acid phosphatase arising from hemolyzed red cells. On the other hand, if lysosomes are considered to be a potential source, the basis for the increased activity of acid phosphatase but not of P-glucuronidase might be explained in terms of differential permeability (53) or of the heterogeneous nature of lysosomes ( R l ) .

ACKNOWLEDGMENTS The author wishes to express his thanks to Miss Anne Reynolds and to Miss Susan London for the typing of this manuscript.

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C7. C8. C9. C10. C11. D1. D2. D3. D4. D5. D6. D7. D8. D9. D10. D11. D12. D13. D14.

Fl. F2.

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